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Fungal pathogens pose significant threats to agricultural crops and food products, leading to economic losses, compromised food quality, and health hazards. Early detection is crucial for effective control and treatment. This study explores Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy for rapid fungal detection in bread. Using a machine learning algorithm (Random Forest), FTIR-ATR accurately distinguished between pure and infected bread samples, achieving 86% overall accuracy and 84% accuracy in identifying specific fungi like Rhizopus and Aspergillus on the first day of infection. These findings highlight FTIR-ATR's potential for early fungal infection detection, promising improved food quality and reduced economic losses through timely intervention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In studying the molecular underpinning of spermatogenesis, we expect to understand the fundamental biological processes better and potentially identify genes that may lead to novel diagnostic and therapeutic strategies toward precision medicine in male infertility. In this review, we emphasized our perspective that the path forward necessitates integrative studies that rely on complementary approaches and types of data. To comprehensively analyze spermatogenesis, this review proposes four axes of integration. First, spanning the analysis of spermatogenesis in the healthy state alongside pathologies. Second, the experimental analysis of model systems (in which we can deploy treatments and perturbations) alongside human data. Third, the phenotype is measured alongside its underlying molecular profiles using known markers augmented with unbiased profiles. Finally, the testicular cells are studied as ecosystems, analyzing the germ cells alongside the states observed in the supporting somatic cells. Recently, the study of spermatogenesis has been advancing using single-cell RNA sequencing, where scientists have uncovered the unique stages of germ cell development in mice, revealing new regulators of spermatogenesis and previously unknown cell subtypes in the testis. An in-depth analysis of meiotic and postmeiotic stages led to the discovery of marker genes for spermatogonia, Sertoli and Leydig cells and further elucidated all the other germline and somatic cells in the testis microenvironment in normal and pathogenic conditions. The outcome of an integrative analysis of spermatogenesis using advanced molecular profiling technologies such as scRNA-seq has already propelled our biological understanding, with additional studies expected to have clinical implications for the study of male fertility. By uncovering new genes and pathways involved in abnormal spermatogenesis, we may gain insights into subfertility or sterility.

The present study aims to identify spermatogenesis in testicular seminiferous tubules (ST) and testicular tissue of adult normal and busulfan-treated mice utilizing PCA and Raman spectroscopy. Raman measurements were conducted on single tubules and testes samples from adult and immature mice, comparing them with those from busulfan-treated adult mice, with validation through histological examination. The analysis revealed a higher signal variability (30 %-40 % at the peaks), prompting scrutiny of individual Raman spectra as a means of spermatogenesis measurement. However, principal component analysis (PCA) demonstrated significant cluster separation between the ST of mature and immature mice. Similar investigations were performed to compare ST from normal mature mice and those from busulfan-treated (BS-treated) mature mice, revealing substantial separation along PC1 and PC2 for all comparison sets. Additionally, comparing testicular samples from mature and immature mice revealed distinct separation in PCA. The study concludes that the combined approach of PCA and Raman spectroscopy proves to be a noninvasive and potentially valuable method for identifying spermatogenesis in seminiferous tubules and testicular samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rapid testing of bacteria for antibiotic susceptibility is essential for effective treatment and curbing the emergence of multidrug-resistant bacteria. The misuse of antibiotics, coupled with the time-consuming classical testing methods, intensifies the threat of antibiotic resistance, a major global health concern. In this study, employing infrared spectroscopy-based machine learning techniques, we significantly shortened the time required for susceptibility testing to 10 hours, a significant improvement from the 24 hours in our previous studies as well as the conventional methods that typically take at least 48 hours. This remarkable reduction in turnaround time (from 48 hours to 10 hours), achieved by minimizing the culturing period, offers a game-changing advantage for clinical applications. Our study involves a dataset comprising 400 bacterial samples (200 E. coli , 100 Klebsiella pneumoniae , and 100 Pseudomonas aeruginosa ) with an impressive 96% accuracy in the taxonomic classification at the species level and up to 82% accuracy in bacterial susceptibility to various antibiotics.

Among the most prevalent and detrimental bacteria causing urinary tract infections (UTIs) is Klebsiella (K.) pneumoniae. A rapid determination of its antibiotic susceptibility can enhance patient treatment and mitigate the spread of resistant strains. In this study, we assessed the viability of using infrared spectroscopy-based machine learning as a rapid and precise approach for detecting K. pneumoniae bacteria and determining its susceptibility to various antibiotics directly from a patient's urine sample. In this study, 2333 bacterial samples, including 636 K. pneumoniae were investigated using infrared micro-spectroscopy. The obtained spectra (27996spectra) were analyzed with XGBoost classifier, achieving a success rate exceeding 95 % for identifying K. pneumoniae. Moreover, this method allows for the simultaneous determination of K. pneumoniae susceptibility to various antibiotics with sensitivities ranging between 74 % and 81 % within approximately 40 min after receiving the patient's urine sample., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gonadotoxic agents could impair spermatogenesis and may lead to male infertility. The present study aimed to evaluate the effect of IL-1β on the development of spermatogenesis from cells isolated from seminiferous tubules (STs) of normal and busulfan-treated immature mice in vitro. Cells were cultured in a 3D in vitro culture system for 5 weeks. We examined the development of cells from the different stages of spermatogenesis by immunofluorescence staining or qPCR analyses. Factors of Sertoli and Leydig cells were examined by qPCR analysis. We showed that busulfan (BU) treatment significantly reduced the expression of testicular IL-1β in the treated mice compared to the control group (CT). Cultures of cells from normal and busulfan-treated immature mice induced the development of pre-meiotic (Vasa), meiotic (Boule), and post-meiotic (acrosin) cells. However, the percentage of developed Boule and acrosin cells was significantly lower in cultures of busulfan-treated mice compared to normal mice. Adding IL-1β to both cultures significantly increased the percentages of Vasa, Boule, and acrosin cells compared to their controls. However, the percentage of Boule and acrosin cells was significantly lower from cultures of busulfan-treated mice that were treated with IL-1β compared to cultures treated with IL-1β from normal mice. Furthermore, addition of IL-1β to cultures from normal mice significantly increased only the expression of androgen receptor and transferrin but no other factors of Sertoli cells compared to their CT. However, the addition of IL-1β to cultures from busulfan-treated mice significantly increased only the expression of androgen-binding protein and the FSH receptor compared to their CT. Adding IL-1β to cultures of normal mice did not affect the expression of 3βHSD compared to the CT, but it significantly reduced its expression in cultures from busulfan-treated mice compared to the CT. Our findings demonstrate the development of different stages of spermatogenesis in vitro from busulfan-treated mice and that IL-1β could potentiate this development in vitro.

Bacteremia refers to the presence of bacteria in the bloodstream, which can lead to a serious and potentially life-threatening condition. In oncology patients, individuals undergoing cancer treatment have a higher risk of developing bacteremia due to a weakened immune system resulting from the disease itself or the treatments they receive. Prompt and accurate detection of bacterial infections and monitoring the effectiveness of antibiotic therapy are essential for enhancing patient outcomes and preventing the development and dissemination of multidrug-resistant bacteria. Traditional methods of infection monitoring, such as blood cultures and clinical observations, are time-consuming, labor-intensive, and often subject to limitations. This manuscript presents an innovative application of infrared spectroscopy of leucocytes of pediatric oncology patients with bacteremia combined with machine learning to diagnose the etiology of infection as bacterial and simultaneously monitor the efficacy of the antibiotic therapy in febrile pediatric oncology patients with bacteremia infections. Through the implementation of effective monitoring, it becomes possible to promptly identify any indications of treatment failure. This, in turn, indirectly serves to limit the progression of antibiotic resistance. The logistic regression (LR) classifier was able to differentiate the samples as bacterial or control within an hour, after receiving the blood samples with a success rate of over 95 %. Additionally, initial findings indicate that employing infrared spectroscopy of white blood cells (WBCs) along with machine learning is viable for monitoring the success of antibiotic therapy. Our follow up results demonstrate an accuracy of 87.5 % in assessing the effectiveness of the antibiotic treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO 2 incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.

Spermatogenesis is the complicated process of sperm generation. During this process, spermatogonial cells proliferate and differentiate via meiotic and post-meiotic stages to produce mature sperm. This process is under the regulation of testicular autocrine/paracrine factors. In addition, endocrine factors are crucial to complete spermatogenesis. We aimed to localize granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR) in testicular cells and further evaluate its involvement in the development of spermatogenesis in vitro. We isolated cells from seminiferous tubule cells of seven-day-old mice and cultured them in vitro using a methylcellulose culture system (MCS), in the presence of GM-CSF and/or testosterone for four weeks. The cells were then examined for markers of different stages of spermatogenesis by immunofluorescence staining and/or qPCR analyses. Our results revealed the presence of GM-CSF and GM-CSFR in testicular cells (premeiotic and meiotic cells as well as somatic cells; Leydig and Sertoli cells). We further demonstrated the development of colonies/spheroids in the MCS which contained pre-meiotic, meiotic, and post-meiotic cells. The addition of GM-CSF to the MCS significantly increased the percentage of pre-meiotic and meiotic cells compared to control. Furthermore, the addition of GM-CSF and testosterone together significantly increased the percentage of cells in the post-meiotic stage compared to the addition of each separately. In conclusion, our results indicate that testicular cells express GM-CSF/GM-CSFR, and that GM-CSF is involved in the development of different stages of spermatogenesis in vitro. Furthermore, testosterone enhances the development of spermatogenic cells and potentiates the effect of GMCSF on the development of post-meiotic cells. These findings provide evidence that GM-CSF and testosterone are involved in the development of spermatogenesis in vitro and in vivo. In brief: Testicular somatic and germ cells express GM-CSF and GM-CSFR. Our study suggests that testicular GM-CSF is involved in the development of spermatogenesis, which is potentiated by testosterone.

Bacterial resistance to antibiotics is a primary global healthcare concern as it hampers the effectiveness of commonly used antibiotics used to treat infectious diseases. The development of bacterial resistance continues to escalate over time. Rapid identification of the infecting bacterium and determination of its antibiotic susceptibility are crucial for optimal treatment and can save lives in many cases. Classical methods for determining bacterial susceptibility take at least 48 h, leading physicians to resort to empirical antibiotic treatment based on their experience. This random and excessive use of antibiotics is one of the most significant drivers of the development of multidrug-resistant (MDR) bacteria, posing a severe threat to global healthcare. To address these challenges, considerable efforts are underway to reduce the testing time of taxonomic classification of the infecting bacterium at the species level and its antibiotic susceptibility determination. Infrared spectroscopy is considered a rapid and reliable method for detecting minor molecular changes in cells. Thus, the main goal of this study was the use of infrared spectroscopy to shorten the identification and the susceptibility testing time of Proteus mirabilis and Pseudomonas aeruginosa from 48 h to approximately 40 min, directly from patients' urine samples. It was possible to identify the Proteus mirabilis and Pseudomonas aeruginosa species with 99% accuracy and, simultaneously, to determine their susceptibility to different antibiotics with an accuracy exceeding 80%.

ἰ-carrageenan is a linear macroalgal polysaccharide that is well known for its antiviral bioactivity. Although it is considered a candidate for antiviral therapeutics, its application is highly limited due to its low solubility and high viscosity, which lower its adsorption efficiency. With the aim of deriving an active ἰ-carrageenan fragment with an improved adsorption capacity, we studied the effects of ultrasonication on structural changes in ἰ-carrageenan with respect to changes in its bioactivity against herpesviruses. An FTIR analysis revealed that ultrasonication increased the hydrophilicity of ἰ-carrageenan without changing its functional groups, and a rheological analysis demonstrated that it gradually decreased the strength of the polysaccharide gel, which completely lost its gel structure and formed small nanoparticles after 30 min of ultrasonication. Concomitantly with these physicochemical changes, a plaque assay revealed that longer ultrasonication increased the antiviral activity of ἰ-carrageenan against two herpesviruses, namely, HSV-1 and VZV. Finally, we separated the 30-min ultrasonicated ἰ-carrageenan into four fractions and found that fractions with a lower molecular weight were significantly less active against both herpesviruses than those with a higher molecular weight. Our findings show that ultrasonication induces physicochemical changes in ἰ-carrageenan that increase its antiviral bioactivity.

Pediatric acute myeloid leukemia (AML) generally occurs de novo. The treatment of AML includes cytarabine (CYT) and other medications. The granulocyte-colony stimulating factor (GCSF) is used in the clinic in cases of neutropenia after chemotherapies. We show that the administration of GCSF in combination with CYT in AML-diagnosed mice (AML+CYT+GCSF) extended the survival of mice for additional 20 days. However, including GCSF in all treatment modalities does not affect the testis' weight or the histology of the seminiferous tubules (STs). We show that GCSF does not affect normal ST histology from AML-, CYT-, or (AML+CYT)-treated groups compared to the relevant treated group without GCSF 2, 4, and 5 weeks post-injection. However, when comparing the percentages of normal STs between the AML+CYT+GCSF-treated groups and those without GCSF, we observe an increase of 17%-42% in STs at 4 weeks and 5.5 weeks post-injection. Additionally, we show that the injection of GCSF into the normal, AML-alone, or CYT-alone groups, or in combination with AML, significantly decreases the percentage of STs with apoptotic cells compared to the relevant groups without GCSF and to the CT (untreated mice) only 2 weeks post-injection. We also show that injection of GCSF into the CT group increases the examined spermatogonial marker PLZF within 2 weeks post-injection. However, GCSF does not affect the count of meiotic cells (CREM) but decreases the post-meiotic cells (ACROSIN) within 4 weeks post-injection. Furthermore, GCSF not only extends the survival of the AML+CYT-treated group, but it also leads to the generation of sperm (1.2 ± 0.04 × 10 6 /mL) at 5.5 weeks post-injection. In addition, we demonstrate that the injection of GCSF into the CT group increases the RNA expression level of IL-10 but not IL-6 compared to CT 2 weeks post-treatment. However, the injection of GCSF into the AML-treated group reverses the expression levels of both IL-10 and IL-6 to normal levels compared to CT 2 weeks post-injection. Thus, we suggest that the addition of GCSF to the regimen of AML after CYT may assist in the development of future therapeutic strategies to preserve male fertility in AML prepubertal patients.

Cancer is the most common and fatal disease around the globe, with an estimated 19 million newly diagnosed patients and approximately 10 million deaths annually. Patients with cancer struggle daily due to difficult treatments, pain, and financial and social difficulties. Detecting the disease in its early stages is critical in increasing the likelihood of recovery and reducing the financial burden on the patient and society. Currently used methods for the diagnosis of cancer are time-consuming, producing discomfort and anxiety for patients and significant medical waste. The main goal of this study is to evaluate the potential of Raman spectroscopy-based machine learning for the identification and characterization of precancerous and cancerous cells. As a representative model, normal mouse primary fibroblast cells (NFC) as healthy cells; a mouse fibroblast cell line (NIH/3T3), as precancerous cells; and fully malignant mouse fibroblasts (MBM-T) as cancerous cells were used. Raman spectra were measured from three different sites of each of the 457 investigated cells and analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA). Our results showed that it was possible to distinguish between the normal and abnormal (precancerous and cancerous) cells with a success rate of 93.1%; this value was 93.7% when distinguishing between normal and precancerous cells and 80.2% between precancerous and cancerous cells. Moreover, there was no influence of the measurement site on the differentiation between the different examined biological systems.

Resistant bacteria have become one of the leading health threats in the last decades. Extended-spectrum β-lactamase (ESBL) producing bacteria, including Escherichia (E.) coli and Klebsiella (K.) pneumoniae (the most frequent ones), are a significant class out of all resistant infecting bacteria. Due to the widespread and ongoing development of ESBL-producing (ESBL + ) resistant bacteria, many routinely used antibiotics are no longer effective against them. However, an early and reliable ESBL + bacteria detection method will improve the efficiency of treatment and limit their spread. In this work, we investigated the capability of infrared (IR) spectroscopy based machine learning tools [principal component analysis (PCA) and Random Forest (RF) classifier] for the rapid detection of ESBL + bacteria isolated directly from patients' urine. For that, we examined 1881 E. coli samples (416 ESBL + and 1465 ESBL - ) and 609 K. pneumoniae samples (237 ESBL + and 372 ESBL - ). All samples were isolated directly from the urine of midstream patients. This study revealed that within 40 min of receiving the patient urine it is possible to determine the infecting bacterium as E. coli or K. pneumoniae with 95% success rate while it was possible to determine the ESBL + E. coli and ESBL + K. pneumoniae with 83% and 78% accuracy rates, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antibiotics are considered the most effective treatment against bacterial infections. However, most bacteria have already developed resistance to a broad spectrum of commonly used antibiotics, mainly due to their uncontrolled use. Extended-spectrum beta-lactamase (ESBL)-producing bacteria are an essential class of multidrug-resistant (MDR) bacteria. It is of extreme urgency to develop a method that can detect ESBL-producing bacteria rapidly for the effective treatment of patients with bacterial infectious diseases. Fourier transform infrared (FTIR) microscopy is a sensitive method that can rapidly detect cellular molecular changes. In this study, we examined the potential of FTIR spectroscopy-based machine learning algorithms for the rapid detection of ESBL-producing bacteria obtained directly from a patient's urine. Using 591 ESBL-producing and 1658 non-ESBL-producing samples of Escherichia coli ( E. coli ) and Klebsiella pneumoniae , our results show that the FTIR spectroscopy-based machine learning approach can identify ESBL-producing bacteria within 40 minutes from receiving a patient's urine sample, with a success rate of 80%.

Bacterial infections cause serious illnesses that are treated with antibiotics. Currently used methods for detecting bacterial antibiotic susceptibility consume 48-72 h, leading to overuse of antibiotics. Thus, many bacterial species have acquired resistance to a broad range of available antibiotics. There is an urgent need to develop efficient methods for rapid determination of bacterial susceptibility to antibiotics. The combination of machine learning and Fourier-transform infrared (FTIR) spectroscopy has generated a promising diagnostic approach in medicine and biology. Our main goal is to examine the potential of FTIR spectroscopy to determine the susceptibility of urinary tract infection-Proteus mirabilis to a specific range of antibiotics, within about 20 min after 24 h culture and identification. We measured the infrared spectra of 489 different P. mirabilis isolates and used random forest to analyze this spectral database. A classification success rate of ~84% was achieved in differentiating between the resistant and sensitive isolates based on their susceptibility to ceftazidime, ceftriaxone, cefuroxime, cefuroxime axetil, cephalexin, ciprofloxacin, gentamicin, and sulfamethoxazole antibiotics in a time span of 24 h instead of 48 h., (© 2022 The Authors. Journal of Biophotonics published by Wiley-VCH GmbH.)

For effective treatment, it is crucial to identify the infecting bacterium at the species level and to determine its antimicrobial susceptibility. This is especially true now, when numerous bacteria have developed multidrug resistance to most commonly used antibiotics. Currently used methods need ∼ 48 h to identify a bacterium and determine its susceptibility to specific antibiotics. This study reports the potential of using infrared spectroscopy with machine learning algorithms to identify E. coli isolated directly from patients' urine while simultaneously determining its susceptibility to antibiotics within ∼ 40 min after receiving the patient's urine sample. For this goal, 1,765 E. coli isolates purified directly from urine samples were collected from patients with urinary tract infections (UTIs). After collection, the samples were tested by infrared microscopy and analyzed by machine learning. We achieved success rates of ∼ 96% in isolate level identification and ∼ 84% in susceptibility determination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Klinefelter syndrome (KS) is the most prevalent genetic disorder of infertile males. This study aimed to determine in Klinefelter patients (KS) the expression levels of spermatogenic markers and testicular growth factors that might predict spermatogenesis based on conventional testicular sperm extraction (TESE). The expression levels of the pre-meiotic (OCT4, CD9, GFR-α1, α-6-INTEGRIN, SALL4, C-KIT), meiotic (CREM-1), and post-meiotic (protamine) markers, as well as the colony stimulating factor-1 (CSF-1) were examined in testicular biopsies with and without mature sperm of KS and normal karyotype of azoospermic patients (AZO) with complete spermatogenesis. In the biopsies of AZO, the expression levels (fold of expression compared to the PPI of the same sample) of OCT4 were 9.68± 7.93, CREM 42.78± 28.22, CSF-1 3.07 ± 3.19, and protamine 78498.12 ± 73214.40. Biopsies from KS included 7 with sperm and 17 without sperm. Among the biopsies with sperm, the expression levels of OCT4 were 7.27± 9.29, CREM 3.13± 7.89, CSF-1 35.5 ± 48.01, and protamine 902.97 ± 2365.92. In 14 biopsies without sperm, we found low expression levels of OCT4, CREM and CSF-1, and no expression of protamine. However, in three of the biopsies without sperm that highly expressed OCT4 and CSF-1, the expression levels of CREM-1 and protamine were high. These results may be used for further consulting with patients considering repeating conventional TESE or micro TESE and cryopreservation for possible future in-vitro spermatogenesis., (© 2022 The Authors. Andrologia published by Wiley-VCH GmbH.)

One of the most common human bacterial infections is the urinary tract infection (UTI). The main cause of UTI is Escherichia ( E .) coli bacteria (∼75%). Because most of the bacteria are resistant to many antibiotics as a result of their indiscriminate overuse, it is extremely important, for effective treatment, to identify the infecting bacteria and to determine, as quickly as possible, their susceptibility to antibiotics. Classical methods require at least 48 hours for determining bacterial susceptibility. In this study, 1798 E. coli isolates from different UTIs were isolated directly from patients' urine, measured by Fourier transform infrared (FTIR) microscopy and analyzed by machine learning algorithms for simultaneous identification and susceptibility determination within 40 minutes since receiving the urine samples. Our results show that it is possible to identify the bacteria at the species level with an accuracy of ∼95% and to determine their susceptibility to different antibiotics with an accuracy ranging from 75% to 83%.

Pseudomonas (P.) aeruginosa is a bacterium responsible for severe infections that have become a real concern in hospital environments. Nosocomial infections caused by P. aeruginosa are often hard to treat because of its intrinsic resistance and remarkable ability to acquire further resistance mechanisms to multiple groups of antimicrobial agents. Thus, rapid determination of the susceptibility of P. aeruginosa isolates to antibiotics is crucial for effective treatment. The current methods used for susceptibility determination are time-consuming; hence the importance of developing a new method. Fourier-transform infra-red (FTIR) spectroscopy is known as a rapid and sensitive diagnostic tool, with the ability to detect minor abnormal molecular changes including those associated with the development of antibiotic- resistant bacteria. The main goal of this study is to evaluate the potential of FTIR spectroscopy together with machine learning algorithms, to determine the susceptibility of P. aeruginosa to different antibiotics in a time span of ∼20 min after the first culture. For this goal, 590 isolates of P. aeruginosa, obtained from different infection sites of various patients, were measured by FTIR spectroscopy and analyzed by machine learning algorithms. We have successfully determined the susceptibility of P. aeruginosa to various antibiotics with an accuracy of 82-90%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Organ/organoid-on-a-chip (OoC) technologies aim to replicate aspects of the in vivo environment in vitro, at the scale of microns. Mimicking the spatial in vivo structure is important and can provide a deeper understanding of the cell-cell interactions and the mechanisms that lead to normal/abnormal function of a given organ. It is also important for disease models and drug/toxin testing. Incorporating active fluid flow in chip models enables many more possibilities. Active flow can provide physical cues, improve intercellular communication, and allow for the dynamic control of the environment, by enabling the efficient introduction of biological factors, drugs, or toxins. All of this is in addition to the fundamental role of flow in supplying nutrition and removing waste metabolites. This review presents an overview of the different types of fluid flow and how they are incorporated in various OoC models. The review then describes various methods and techniques of incorporating perfusion networks into OoC models, including self-assembly, bioprinting techniques, and utilizing sacrificial gels. The second part of the review focuses on the replication of spermatogenesis in vitro; the complex process whereby spermatogonial stem cells differentiate into mature sperm. A general overview is given of the various approaches that have been used. The few studies that incorporated microfluidics or vasculature are also described. Finally, a future perspective is given on elements from perfusion-based models that are currently used in models of other organs and can be applied to the field of in vitro spermatogenesis. For example, adopting tubular blood vessel models to mimic the morphology of the seminiferous tubules and incorporating vasculature in testis-on-a-chip models. Improving these models would improve our understanding of the process of spermatogenesis. It may also potentially provide novel therapeutic strategies for pre-pubertal cancer patients who need aggressive chemotherapy that can render them sterile, as well asfor a subset of non-obstructive azoospermic patients with maturation arrest, whose testes do not produce sperm but still contain some of the progenitor cells.

This research presents a novel testis-on-a-chip (ToC) platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into spheroids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed spheroids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The spheroid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment was also investigated. As a benchmark, the ToC is compared to a conventional three-dimensional methylcellulose cell culture system in a well plate. Analysis via fluorescence-activated cell sorting shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesis in-vitro . It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre-pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing., (© 2022 IOP Publishing Ltd.)

Acute myeloid leukemia (AML) accounts for around 20% of diagnosed childhood leukemia. Cytarabine (CYT) is involved in the AML treatment regimen. AML and CYT showed impairment in spermatogenesis in human and rodents in adulthood. We successfully developed an AML disease model in sexually immature mice. Monocytes and granulocytes were examined in all groups: untreated control, AML alone, CYT alone and AML+CYT (in combination). There was a significant increase in the counts of monocytes and granulocytes in the AML-treated immature mice (AML) compared to the control, and AML cells were demonstrated in the blood vessels of the testes. AML alone and CYT alone impaired the development of spermatogenesis at the adult age of the AML-treated immature mice. The damage was clear in the structure/histology of their seminiferous tubules, and an increase in the apoptotic cells of the seminiferous tubules was demonstrated. Our results demonstrated a significant decrease in the meiotic/post-meiotic cells compared to the control. However, CYT alone (but not AML) significantly increased the count of spermatogonial cells (premeiotic cells) that positively stained with SALL4 and PLZF per tubule compared to the control. Furthermore, AML significantly increased the count of proliferating spermatogonial cells that positively stained with PCNA in the seminiferous tubules compared to the control, whereas CYT significantly decreased the count compared to the control. Our result showed that AML and CYT affected the microenvironment/niche of the germ cells. AML significantly decreased the levels growth factors, such as SCF, GDNF and MCSF) compared to control, whereas CYT significantly increased the levels of MCSF and GDNF compared to control. In addition, AML significantly increased the RNA expression levels of testicular IL-6 (a proinflammatory cytokine), whereas CYT significantly decreased testicular IL-6 levels compared to the control group. Furthermore, AML alone and CYT alone significantly decreased RNA expression levels of testicular IL-10 (anti-inflammatory cytokine) compared to the control group. Our results demonstrate that pediatric AML disease with or without CYT treatment impairs spermatogenesis at adult age (the impairment was more pronounced in AML+CYT) compared to control. Thus, we suggest that special care should be considered for children with AML who are treated with a CYT regimen regarding their future fertility at adult age.

Infertility affects one in six couples, half of which are caused by a male factor. Male infertility can be caused by both, qualitative and quantitative defects, leading to Oligo- astheno-terato-zoospermia (OAT; impairment in ejaculate sperm cell concentration, motility and morphology). Azoospermia defined as complete absence of sperm cells in the ejaculation. While hundreds of genes are involved in spermatogenesis the genetic etiology of men's infertility remains incomplete.We identified a hemizygous stop gain pathogenic variation (PV) in the X-linked Germ Cell Nuclear Acidic Peptidase (GCNA), in an Azoospermic patient by exome sequencing. Assessment of the prevalence of pathogenic variations in this gene in infertile males by exome sequence data of 11 additional unrelated patients identified a probable hemizygous causative missense PV in GCNA in a severe OAT patient. Expression of GCNA in the patients' testes biopsies and the stage of spermatogonial developmental arrest were determined by immunofluorescence and immunohistochemistry. The Azoospermic patient presented spermatogenic maturation arrest with an almost complete absence of early and late primary spermatocytes and thus the complete absence of sperm. GCNA is critical for genome integrity and its loss results in genomic instability and infertility in Drosophila, C. elegans, zebrafish, and mouse. PVs in GCNA appear to be incompatible with male fertility in humans as well: A stop-gain PV caused Azoospermia and a missense PV caused severe OAT with very low fertilization rates and no pregnancy in numerous IVF treatments., (© 2021. The Author(s), under exclusive licence to European Society of Human Genetics.)

Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.

Klebsiella pneumoniae ( K. pneumoniae) is one of the most aggressive multidrug-resistant bacteria associated with human infections, resulting in high mortality and morbidity. We obtained 1190 K. pneumoniae isolates from different patients with urinary tract infections. The isolates were measured to determine their susceptibility regarding nine specific antibiotics. This study's primary goal is to evaluate the potential of infrared spectroscopy in tandem with machine learning to assess the susceptibility of K. pneumoniae within approximately 20 min following the first culture. Our results confirm that it was possible to classify the isolates into sensitive and resistant with a success rate higher than 80% for the tested antibiotics. These results prove the promising potential of infrared spectroscopy as a powerful method for a K. pneumoniae susceptibility test.

Background: Mutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum ( SCAPER ) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies., Methods: Human clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of short spindle 3 ( ssp3 ) ( SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy., Results: We show that patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells . Interestingly, Drosophil a null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. We further show that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs., Conclusions: Our results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Spermatogenesis is a complex process, in which spermatogonial cells proliferate and differentiate in the seminiferous tubules of the testis to generate sperm. This process is under the regulation of endocrine and testicular paracrine/autocrine factors. In the present study, we demonstrated that colony stimulating factor-1 (CSF-1) is produced by mouse testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells (such as CDH1-positively stained cells; a marker of spermatogonial cells). In addition, we demonstrated the presence of CSF-1 and its receptor (CSF-1R) in testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells of human testis. We also show that the protein levels of CSF-1 were the highest in testis of 1-week-old mice and significantly decreased with age (2-12-week-old). However, the transcriptome levels of CSF-1 significantly increased in 2-3-week-old compared to 1-week-old, and thereafter significantly decreased with age. On the other hand, the transcriptome levels of CSF-1R was significantly higher in mouse testicular tissue of all examined ages (2-12-week-old) compared to 1-week-old. Our results demonstrate the involvement of CSF-1 in the induction the proliferation and differentiation of spermatogonial cells to meiotic and postmeiotic stages (BOULE- and ACROSIN-positive cells) under in vitro culture conditions, using methylcellulose culture system (MCS). Thus, it is possible to suggest that CSF-1 system, as a testicular paracrine/autocrine system, is involved in the development of different stages of spermatogenesis and may be used in the development of future therapeutic strategies for treatment of male infertility.

Antimicrobial drugs have played an indispensable role in decreasing morbidity and mortality associated with infectious diseases. However, the resistance of bacteria to a broad spectrum of commonly-used antibiotics has grown to the point of being a global health-care problem. One of the most important classes of multi-drug resistant bacteria is Extended Spectrum Beta-Lactamase-producing (ESBL+) bacteria. This increase in bacterial resistance to antibiotics is mainly due to the long time (about 48 h) that it takes to obtain lab results of detecting ESBL-producing bacteria. Thus, rapid detection of ESBL+ bacteria is highly important for efficient treatment of bacterial infections. In this study, we evaluated the potential of infrared microspectroscopy in tandem with machine learning algorithms for rapid detection of ESBL-producing Klebsiella pneumoniae (K. pneumoniae) obtained from samples of patients with urinary tract infections. 285 ESBL+ and 365 ESBL-K. pneumoniae samples, gathered from cultured colonies, were examined. Our results show that it is possible to determine that K. pneumoniae is ESBL+ with ∼89% accuracy, ∼88% sensitivity and ∼89% specificity, in a time span of ∼20 minutes following the initial culture.

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions.

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis., Competing Interests: The authors declare no conflict of interest.

Spermatogenesis is the process of spermatogonial stem cell (SSC) proliferation and differentiation to generate sperm. This process is regulated by cell-cell interactions between Sertoli cells and developing SSCs by autocrine/paracrine and endocrine factors. It is also affected by cells in the interstitial compartment, such as Leydig cells and peritubular cells. Here, we demonstrate, for the first time, the presence of interleukin-34 (IL-34) in Leydig, Sertoli, and peritubular cells and in the premeiotic, meiotic, and postmeiotic cells. Its receptor, colony-stimulating factor-1 (CSF-1), has already been demonstrated in Leydig, Sertoli, premeiotic, and meiotic cells. IL-34 was detected in testicular homogenates and Sertoli cell-conditioned media, and was affected by mouse age. We showed that the addition of IL-34 in vitro to isolated cells from the seminiferous tubules of 7-day-old mice, using the methylcellulose culture system (MCS), increased the percentages and expression of the premeiotic cells (VASA), the meiotic cells (BOULE), and the meiotic/postmeiotic cells (ACROSIN) after four weeks of culture, when examined by immunofluorescence staining (IF) and qPCR analysis. It is possible to suggest that IL-34 is a novel paracrine/autocrine factor involved in the development of spermatogenesis. This factor may be used in future therapeutic strategies for the treatment of male infertility.

Physicians diagnose subjectively the etiology of inaccessible infections where sampling is not feasible (such as, pneumonia, sinusitis, cholecystitis, peritonitis), as bacterial or viral. The diagnosis is based on their experience with some medical markers like blood counts and medical symptoms since it is harder to obtain swabs and reliable laboratory results for most cases. In this study, infrared spectroscopy with machine learning algorithms was used for the rapid and objective diagnosis of the etiology of inaccessible infections and enables an assessment of the error for the subjective diagnosis of the etiology of these infections by physicians. Our approach allows for diagnoses of the etiology of both accessible and inaccessible infections as based on an analysis of the innate immune system response through infrared spectroscopy measurements of white blood cell (WBC) samples. In the present study, we examined 343 individuals involving 113 controls, 89 inaccessible bacterial infections, 54 accessible bacterial infections, 60 inaccessible viral infections, and 27 accessible viral infections. Using our approach, the results show that it is possible to differentiate between controls and infections (combined bacterial and viral) with 95% accuracy, and enabling the diagnosis of the etiology of accessible infections as bacterial or viral with >94% sensitivity and > 90% specificity within one hour after the collection of the blood sample with error rate <6%. Based on our approach, the error rate of the physicians' subjective diagnosis of the etiology of inaccessible infections was found to be >23%.

Male fertility preservation is required when treatment with an aggressive chemo-/-radiotherapy, which may lead to irreversible sterility. Due to new and efficient protocols of cancer treatments, surviving rates are more than 80%. Thus, these patients are looking forward to family life and fathering their own biological children after treatments. Whereas adult men can cryopreserve their sperm for future use in assistance reproductive technologies (ART), this is not an option in prepubertal boys who cannot produce sperm at this age. In this review, we summarize the different technologies for male fertility preservation with emphasize on prepubertal, which have already been examined and/or demonstrated in vivo and/or in vitro using animal models and, in some cases, using human tissues. We discuss the limitation of these technologies for use in human fertility preservation. This update review can assist physicians and patients who are scheduled for aggressive chemo-/radiotherapy, specifically prepubertal males and their parents who need to know about the risks of the treatment on their future fertility and the possible present option of fertility preservation., Competing Interests: The authors declare no conflict of interest.

Background: Ubiquitin-Specific Peptidase 26 (USP26), located on the X chromosome, encodes a deubiquitinating enzyme expressed mainly in testis, where it regulates protein turnover during spermatogenesis and modulates the ubiquitination levels of the Androgen Receptor (AR), and as a consequence, affects AR signaling., Methods: The patient was thoroughly characterized clinically. He was genetically tested by chromosome analysis and whole exome sequencing (WES)., Results: The patient was diagnosed with Sertoli cell-only syndrome pattern (SCOS). The WES analysis revealed only the variation in USP26: causing p.P469S in a highly evolutionary conserved amino acid as the possible cause for SCOS. The literature search identified 34 single variations and 14 clusters of variations in USP26 that were associated with male infertility. Only one of the 22 variations and of one cluster of three mutations tested for ubiquitination activity was found as damaging. Only one out of six variations tested for effect on AR function was found as damaging. Thus, the association of USP26 with male fertility was questioned., Conclusions: The finding in our patient and the discussion on the reviewed literature support a possible role for USP26 in male fertility., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

HTLV-1 is a human retrovirus responsible for adult T-cell leukemia (ATL) and certain other clinical disorders. The viral Tax oncoprotein plays a central role in HTLV-1 pathogenicity, mainly due to its capacity of inducing the transcriptional activity of various transcriptional factors like NFқB. Eucalyptus camaldulensis ( Ec ) is considered as a traditional medicinal plant with valuable therapeutic effects. Here we evaluated the activity of its ethanolic leave extract on different Tax activities by testing its influence on Tax-induced activity of NFқB and HTLV-1 LTR in Jurkat cells. Our results showed that Ec inhibited Tax induced activation of NFқB -, SRF- dependent promoters and HTLV-1 LTR. Ec extract has no effect on the binding of Tax to NFқB while it strongly prevented the degradation of IҝBα induced by Tax probably as a result of preventing the link between Tax and IKKγ. In addition, increasing the cellular level of P-TEFb-cyclinT1 significantly reduced the inhibitory effect of Ec on Tax activities, probably by preventing the interaction between Tax and P-TEFb-cyclin T1. The 40%-MeOH fraction of this extract, which is rich with polyphenols, offered the highest inhibitory effect against Tax activities. Further studies are required for the isolation and identification of active component/s in this extract which may be developed in the future as preventive/curing drugs for HTLV-1 related diseases.

Study Question: Can a machine-learning-based model trained in clinical and biological variables support the prediction of the presence or absence of sperm in testicular biopsy in non-obstructive azoospermia (NOA) patients?, Summary Answer: Our machine-learning model was able to accurately predict (AUC of 0.8) the presence or absence of spermatozoa in patients with NOA., What Is Known Already: Patients with NOA can conceive with their own biological gametes using ICSI in combination with successful testicular sperm extraction (TESE). Testicular sperm retrieval is successful in up to 50% of men with NOA. However, to the best of our knowledge, there is no existing model that can accurately predict the success of sperm retrieval in TESE. Moreover, machine-learning has never been used for this purpose., Study Design, Size, Duration: A retrospective cohort study of 119 patients who underwent TESE in a single IVF unit between 1995 and 2017 was conducted. All patients with NOA who underwent TESE during their fertility treatments were included. The development of gradient-boosted trees (GBTs) aimed to predict the presence or absence of spermatozoa in patients with NOA. The accuracy of these GBTs was then compared to a similar multivariate logistic regression model (MvLRM)., Participants/materials, Setting, Methods: We employed univariate and multivariate binary logistic regression models to predict the probability of successful TESE using a dataset from a retrospective cohort. In addition, we examined various ensemble machine-learning models (GBT and random forest) and evaluated their predictive performance using the leave-one-out cross-validation procedure. A cutoff value for successful/unsuccessful TESE was calculated with receiver operating characteristic (ROC) curve analysis., Main Results and the Role of Chance: ROC analysis resulted in an AUC of 0.807 ± 0.032 (95% CI 0.743-0.871) for the proposed GBTs and 0.75 ± 0.052 (95% CI 0.65-0.85) for the MvLRM for the prediction of presence or absence of spermatozoa in patients with NOA. The GBT approach and the MvLRM yielded a sensitivity of 91% vs. 97%, respectively, but the GBT approach has a specificity of 51% compared with 25% for the MvLRM. A total of 78 (65.3%) men with NOA experienced successful TESE. FSH, LH, testosterone, semen volume, age, BMI, ethnicity and testicular size on clinical evaluation were included in these models., Limitations, Reasons for Caution: This study is a retrospective cohort study, with all the associated inherent biases of such studies. This model was used only for TESE, since micro-TESE is not performed at our center., Wider Implications of the Findings: Machine-learning models may lay the foundation for a decision support system for clinicians together with their NOA patients concerning TESE. The findings of this study should be confirmed with further larger and prospective studies., Study Funding/competing Interest(s): The study was funded by the Division of Obstetrics and Gynecology, Soroka University Medical Center, there are no potential conflicts of interest for all authors., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pectobacterium and Dickeya spp. are soft rot Pectobacteriaceae that cause aggressive diseases on agricultural crops leading to substantial economic losses. The accurate, rapid and low-cost detection of these pathogenic bacteria are very important for controlling their spread, reducing the consequent financial loss and for producing uninfected potato seed tubers for future generations. Currently used methods for the identification of these bacterial pathogens at the strain level are based mainly on molecular techniques, which are expensive. We used an alternative method, infrared spectroscopy, to measure 24 strains of five species of Pectobacterium and Dickeya. Measurements were then analyzed using machine learning methods to differentiate among them at the genus, species and strain levels. Our results show that it is possible to differentiate among different bacterial pathogens with a success rate of ~99% at the genus and species levels and with a success rate of over 94% at the strain level., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rapid diagnosis of the etiology of infection is highly important for an effective treatment of the infected patients. Bacterial and viral infections are serious diseases that can cause death in many cases. The human immune system deals with many viral and bacterial infections that cause no symptoms and pass quietly without treatment. However, oncology patients undergoing chemotherapy have a very weak immune system caused by leukopenia, and even minor pathogen infection threatens their lives. For this reason, physicians tend to prescribe immediately several types of antibiotics for febrile pediatric oncology patients (FPOPs). Uncontrolled use of antibiotics is one of the major contributors to the development of resistant bacteria. Therefore, for oncology patients, a rapid and objective diagnosis of the etiology of the infection is extremely critical. Current identification methods are time-consuming (>24 h). In this study, the potential of midinfrared spectroscopy in tandem with machine learning algorithms is evaluated for rapid and objective diagnosis of the etiology of infections in FPOPs using simple peripheral blood samples. Our results show that infrared spectroscopy enables the diagnosis of the etiology of infection as bacterial or viral within 70 minutes after the collection of the blood sample with 93% sensitivity and 88% specificity., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although several effective treatments exist against Varicella zoster virus (VZV), resistant strains have emerged and the treatment is usually not definite and may have various undesired side effects. Thus, alternative treatment options are necessary. Here we studied the inhibitory effects of natural polysaccharides (PSs) obtained from renewable sources, varied by their structure and charge, on VZV infection in vitro , using a plaque assay. In terms of selectivity indices, almost all the tested PSs were very active; in the order of λ  >  ἰ  >  G >  κ  >  P against VZV compared to Acyclovir as a reference drug and exhibited dose-dependent behavior. Our results, which showed a strong inhibition of VZV infection when the cells were treated with ἰ only at the time of infection or only post infection may indicate a multistep inhibitory effect. It seems that ἰ may block different stages of the virus replication cycle including early steps such as absorption and penetration to the host cells and also late steps after the penetration into the host cells. These results are part of an on-going research that highlights the PSs as potential novel nontoxic candidates that can be used against VZV, and contributes to the elucidation of their mode of action.

Objective: To elucidate the potential regulatory function of miR-23a/b-3p on spermatogenesis-specific genes., Design: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation, Northern blot, dual luciferase assay, and Western blot confirmation., Setting: University research and clinical institutes., Patient(s): A total of 115 men presenting at an infertility clinic., Intervention(s): None., Main Outcome Measure(s): Significant higher abundance levels of miR-23a/b-3p and lower abundance levels of PFKFB4, HMMR, SPATA6, and TEX15 in oligoasthenozoospermic men compared with those in normozoospermic men., Result(s): In oligoasthenozoospermic men, the abundance levels of miR-23a/b-3p were significantly higher when compared with controls as determined by RT-qPCR. After in silico prediction of potential targets of miR-23a/b-3p, PFKFB4, HMMR, SPATA6, and TEX15 have been identified as direct targets by dual luciferase assays. Mutations in the miR-23a/b-3p binding site within the 3'UTRs resulted in abrogated responsiveness to miR-23a/b-3p. PFKFB4, HMMR, SPATA6, and TEX15 mRNA and HMMR and SPATA6 protein levels were significantly lower in oligoasthenozoospermic men compared with in normozoospermic men. Correlation analysis showed that the sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with PFKFB4, HMMR, SPATA6, and TEX15 abundance levels (lower ΔCt, the higher abundance levels)., Conclusion(s): This study establishes a link between up-regulation of miR-23a/b-3p and the coincident down-regulation of four expressed genes in the sperm of men with oligoasthenozoospermia, compared with men with normozoospermia. This study provides a novel insight into some of the mechanisms leading to male subfertility, offering a possible therapeutic target for treatment, or even for male contraception., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Antimicrobial drugs have an important role in controlling bacterial infectious diseases. However, the increasing resistance of bacteria to antibiotics has become a global health care problem. Rapid determination of antimicrobial susceptibility of clinical isolates is often crucial for the optimal antimicrobial therapy. The conventional methods used in medical centers for susceptibility testing are time-consuming (>2 days). Two bacterial culture steps are needed, the first is used to grow the bacteria from urine on agar plates to determine the species of the bacteria (~24 hours). The second culture is used to determine the susceptibility by growing colonies from the first culture for another 24 hours. Here, the main goal is to examine the potential of infrared microscopy combined with multivariate analysis, to reduce the time it takes to identify Escherichia coli susceptibility to antibiotics and to determine the optimum choice of antibiotic to which the bacteria will respond. E coli colonies of the first culture from patients with urinary tract infections (UTI) were examined for the bacterial susceptibility using Fourier-transform infrared (FTIR). Our results show that it is possible to determine the optimum choice of antibiotic with better than 89% sensitivity, in the time span of few minutes, following the first culture., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Spermatogenesis is the process of the proliferation and differentiation of spermatogonial stem cells (SSCs) to generate sperm. Leukemia patients show impairment in some of the endocrine hormones that are involved in spermatogenesis. They also show a decrease in semen parameters before and after thawing of cryopreserved samples compared to a control. The mechanisms behind these effects have not yet been described. This review summarizes the effect of leukemia on semen parameters from adult patients and highlights feasible suggested mechanisms that may affect impairment of spermatogenesis in these patients. We suggest the possible involvement of leukemia in disturbing hormones involved in spermatogenesis, and the imbalance in testicular paracrine/autocrine factors involved in the formation of SSC niches that control their proliferation and differentiation. Understanding the mechanisms of leukemia in the impairment of spermatogenesis may lead to the development of novel therapeutic strategies mainly for prepubertal boys who do not yet produce sperm.

The spread of multidrug resistant bacteria has become a global concern. One of the most important and emergent classes of multidrug-resistant bacteria is extended-spectrum β-lactamase-producing bacteria (ESBL-positive = ESBL + ). Due to widespread and continuous evolution of ESBL-producing bacteria, they become increasingly resistant to many of the commonly used antibiotics, leading to an increase in the mortality associated with resulting infections. Timely detection of ESBL-producing bacteria and rapid determination of their susceptibility to appropriate antibiotics can reduce the spread of these bacteria and the consequent complications. Routine methods used for the detection of ESBL-producing bacteria are time-consuming, requiring at least 48 h to obtain results. In this study, we evaluated the potential of infrared spectroscopic microscopy, combined with multivariate analysis for rapid detection of ESBL-producing Escherichia coli ( E. coli) isolated from urinary-tract infection (UTI) samples. Our measurements were conducted on 837 samples of uropathogenic E. coli (UPEC), including 268 ESBL + and 569 ESBL-negative (ESBL - ) samples. All samples were obtained from bacterial colonies after 24 h culture (first culture) from midstream patients' urine. Our results revealed that it is possible to detect ESBL-producing bacteria, with a 97% success rate, 99% sensitivity, and 94% specificity for the tested samples, in a time span of few minutes following the first culture.

Sertoli cell-only syndrome (SCOS) affects about 26.3⁻57.8% of azoospermic men, with their seminiferous tubules containing only Sertoli cells. Recently, it was reported that testicular biopsies from nonobstructive azoospermic (NOA) patients contained germ cells, and that sperm could be found in the tubules of 20% of SCOS patients using testicular sperm extraction technology. Since the patients without sperm in their testicular biopsies do not have therapy to help them to father a biological child, in vitro maturation of spermatogonial stem cells (SSCs) isolated from their testis is a new approach for possible future infertility treatment. Recently, the induction of human and mice SSCs proliferation and differentiation was demonstrated using different culture systems. Our group reported the induction of spermatogonial cell proliferation and differentiation to meiotic and postmeiotic stages in mice, rhesus monkeys, and prepubertal boys with cancer using 3D agar and methylcellulose (MCS) culture systems. The aim of the study was to identify the type of spermatogenic cells present in biopsies without sperm from SCOS patients, and to examine the possibility of inducing spermatogenesis from isolated spermatogonial cells of these biopsies in vitro using 3D MCS. We used nine biopsies without sperm from SCOS patients, and the presence of spermatogenic markers was evaluated by PCR and specific immunofluorescence staining analyses. Isolated testicular cells were cultured in MCS in the presence of StemPro enriched media with different growth factors and the development of colonies/clusters was examined microscopically. We examined the presence of cells from the different stages of spermatogenesis before and after culture in MCS for 3⁻7 weeks. Our results indicated that these biopsies showed the presence of premeiotic markers (two to seven markers/biopsy), meiotic markers (of nine biopsies, cAMP responsive element modulator-1 (CREM-1) was detected in five, lactate dehydrogenase (LDH) in five, and BOULE in three) and postmeiotic markers (protamine was detected in six biopsies and acrosin in three). In addition, we were able to induce the development of meiotic and/or postmeiotic stages from spermatogonial cells isolated from three biopsies. Thus, our study shows for the first time the presence of meiotic and/or postmeiotic cells in biopsies without the sperm of SCOS patients. Isolated cells from some of these biopsies could be induced to meiotic and/or postmeiotic stages under in vitro culture conditions.

Leukemia is one of the most common cancers in patients of reproductive age. It is well known that chemotherapy, used as anti-cancer therapy, adversely affects male fertility. Moreover, the negative effect of leukemia on sperm quality, even before chemotherapy treatment, has been reported. However, the mechanisms behind this disease's effect on sperm quality remains unknown. In this study, we examine the direct effect of leukemia and chemotherapy alone and in combination on sperm parameters and male fertility. For this, we developed an acute myeloid leukemia (AML) mouse model (mice were treated with AML cells C1498 and developed leukemia); these mice then received cytarabine chemotherapy. Our findings reveal a significant reduction in sperm concentration and motility and a significant increase in abnormal morphology and spontaneous acrosome reaction of the sperm following AML and chemotherapy treatment, alone and in combination. We also found a reduction in male fertility and the number of delivered offspring. Our results support previous findings that AML impairs sperm parameters and show for the first time that AML increases spontaneous acrosome reaction and decreases male fertility capacity and number of offspring.

Hao Win, Hui Ma and Sajjad Hussain were incorrectly affiliated to 'Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX 77030 USA'. These authors should only have been affiliated to 'Hefei National Laboratory for Physical Sciences at Microscale, The First Affiliated Hospital of USTC, USTC-SJH Joint Center for Human Reproduction and Genetics, The CAS Key Laboratory of Innate Immunity and Chronic Diseases, School of Life Sciences, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center of Genetics and Development, University of Science and Technology of China, Hefei 230027, China'. They were also not noted as contributing equally to the paper. Both these errors have now been corrected in the PDF and HTML versions of the paper.