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The Kirsten Rat Sarcoma Virus (KRAS) oncoprotein, one of the most prevalent mutations in cancer, has been deemed undruggable for decades. The hypothesis of this work was that delivering anti-KRAS monoclonal antibody (mAb) at the intracellular level could effectively target the KRAS oncoprotein. To reach this goal, we designed and developed tLyP1-targeted palmitoyl hyaluronate (HAC16)-based nanoassemblies (HANAs) adapted for the association of bevacizumab as a model mAb. Selected candidates with adequate physicochemical properties (below 150 nm, neutral surface charge), and high drug loading capacity (>10%, w /w) were adapted to entrap the antiKRASG12V mAb. The resulting antiKRASG12V-loaded HANAs exhibited a bilayer composed of HAC16 polymer and phosphatidylcholine (PC) enclosing a hydrophilic core, as evidenced by cryogenic-transmission electron microscopy (cryo-TEM) and X-ray photoelectron spectroscopy (XPS). Selected prototypes were found to efficiently engage the target KRASG12V and, inhibit proliferation and colony formation in KRASG12V-mutated lung cancer cell lines. In vivo , a selected formulation exhibited a tumor growth reduction in a pancreatic tumor-bearing mouse model. In brief, this study offers evidence of the potential to use nanotechnology for developing anti-KRAS precision therapy and provides a rational framework for advancing mAb intracellular delivery against intracellular targets. [Display omitted] • The HANAs technology represents a new strategy for the intracellular delivery of mAbs and, notably, antiKRASG12V mAb. • The HANAs' surface can be functionalized with the tumor-penetrating peptide tLyP1. • The antiKRASG12V mAb-HANAs technology was able to engage the KRASG12V oncoprotein and exhibit a significant reduction in tumor growth in vivo. [ABSTRACT FROM AUTHOR]

Senescent cells produce a Senescence‐Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin‐6 (IL‐6), has the potential to amplify cellular senescence in the SASP‐producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL‐6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL‐6 sustains intracrine senescence interacting with the intracellular IL‐6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS‐STING, and NFκB activation. This pathway triggered by intracellular IL‐6 significantly contributes to cell‐autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL‐6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re‐expression of IL‐6 restores senescence control of tumor growth. The intracrine senescent IL‐6 pathway is further evidenced in three human cellular models of therapy‐induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL‐6 to sustain cell‐autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence‐based therapies. [ABSTRACT FROM AUTHOR]

Clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae, leads to the formation of galls, commonly known as pathogen-induced tumors, on the roots of infected plants. The identification of crucial regulators of host tumor formation is essential to unravel the mechanisms underlying the proliferation and differentiation of P. brassicae within plant cells. To gain insight into this process, transcriptomic analysis was conducted to identify key genes associated with both primary and secondary infection of P. brassicae in Chinese cabbage. Our results demonstrate that the k-means clustering of subclass 1, which exhibited specific trends, was closely linked to the infection process of P. brassicae. Of the 1610 differentially expressed genes (DEGs) annotated in subclass 1, 782 were identified as transcription factors belonging to 49 transcription factor families, including bHLH, B3, NAC, MYB_related, WRKY, bZIP, C2H2, and ERF. In the primary infection, several genes, including the predicted Brassica rapa probable pectate lyase, RPM1-interacting protein 4-like, L-type lectin-domain-containing receptor kinase, G-type lectin S-receptor-like serine, B. rapa photosystem II 22 kDa protein, and MLP-like protein, showed significant upregulation. In the secondary infection stage, 45 of 50 overlapping DEGs were upregulated. These upregulated DEGs included the predicted B. rapa endoglucanase, long-chain acyl-CoA synthetase, WRKY transcription factor, NAC domain-containing protein, cell division control protein, auxin-induced protein, and protein variation in compound-triggered root growth response-like and xyloglucan glycosyltransferases. In both the primary and secondary infection stages, the DEGs were predicted to be Brassica rapa putative disease resistance proteins, Ltype lectin domain-containing receptor kinases, ferredoxin-NADP reductases, 1-aminocyclopropane-1-carboxylate synthases, histone deacetylases, UDPglycosyltransferases, putative glycerol-3-phosphate transporters, and chlorophyll a-binding proteins, which are closely associated with plant defense responses, biosynthetic processes, carbohydrate transport, and photosynthesis. This study revealed the pivotal role of transcription factors in the initiation of infection and establishment of intracellular parasitic relationships during the primary infection stage, as well as the proliferation and differentiation of the pathogenwithin the host cell during the secondary infection stage. [ABSTRACT FROM AUTHOR]

Methylorubrum extorquens DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derivedmethanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont M. extorquens DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (phaC), PHB depolymerase (phaZ1), and a transcription factor (phaR) showed altered PHB granule characteristics, as ΔphaC had a significantly low number of granules, DphaR had a significantly increased number of granules, and DphaZ1 had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the DphaC strain was sensitive to 10 mM HO· and 20 mM H2O2. The colonization of the host, Scots pine (Pinus sylvestris L.), by the deletion strains varied greatly. The deletion strain ΔphaR colonized the host mainly intercellularly, whereas the ΔphaZ1 strain was a slightly poorer colonizer than the control. The deletion strain ΔphaC lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by ΔhaC and ΔhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont M. extorquens DSM13060. Our current results show that low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in M. extorquens DSM13060 is an important trait in the non-rhizobial endosymbiosis. [ABSTRACT FROM AUTHOR]

Polyhydroxybutyrate (PHB) is known for wide applications, biocompatibility, and degradability; however, it cannot be commercialized due to conventional recovery using solvents. The present study employed mechanical cell-disruption methods, such as Pestle and mortar, sonication, and glass bead vortexing, for solvent-free extraction of PHA from Bacillus sp. Different time intervals were set for grinding (5, 10, 15 min), sonicating (1, 3 and 5 min), and vortexing (2, 5 and 8 g glass beads with 5, 10 and 15 min each) hence studying their effect on cell lysis to release PHA. Tris buffer containing phenylmethyl sulfonyl fluoride (PMSF) (20 mM Tris–HCl, pH 8.0, 1 mM PMSF) was employed as a lysis buffer to study its action over Bacillus cells. Its presence was checked with the above methods in cell lysis. Sonicating cells for 5 min in the presence of lysis buffer achieved a maximum PHA yield of 45%. Cell lysis using lysis buffer yielded 35% PHA when vortexing with 5 g glass beads for 15 min. Grinding cells for 15 min showed a maximum yield of 34% but lacked a lysis buffer. The overall results indicated that the action of lysis buffer and physical extraction methods improved PHA yield by %. Therefore, the study sought to evaluate the feasibility of applying laboratory methods for cell disruption. These methods can showcase possible opportunities in large-scale applications. The polymer yield results were compared with standard sodium hypochlorite extraction. Confirmation of obtained polymers as polyhydroxy butyrate (PHB) was made through FTIR and 1HNMR characterization. [ABSTRACT FROM AUTHOR]

Connexin is a transmembrane protein present on the cell membrane of most cell types. Connexins assemble into a hexameric hemichannel known as connexon that pairs with another hemichannel present on a neighboring cell to form gap junction that acts as a channel or pore for the transport of ions and small molecules between the cytoplasm of the two cells. Extracellular vesicles released from connexin-expressing cells could carry connexin hemichannels on their surface and couple with another connexin hemichannel on a distant recipient cell to allow the transfer of the intravesicular content directly into the cytoplasm. Connexin-containing vesicles can be potentially utilized for intracellular drug delivery. In this review, we introduced cell-derived, connexin-containing extracellular vesicles and cell-free connexin-containing liposomes, methods of preparing them, procedures to load cargos in them, factors regulating the connexin hemichannel activity, (potential) applications of connexin-containing vesicles in drug delivery, and finally the challenges and future directions in realizing the promises of this platform delivery system for (intracellular) drug delivery. [ABSTRACT FROM AUTHOR]

The members of genus Acanthamoeba are the etiological agent of uncommon but severe or even fatal opportunistic infections in human beings. The presence of different classes of intracellular and extracellular proteases including serine proteases, cysteine proteases, and metalloproteases has been well documented in environmental and clinical isolates of Acanthamoeba spp. However, the role of the proteolytic enzymes in physiological, biological, and pathological mechanisms of the amoeba remains partially investigated. Some attempts have been conducted using various methods to determine the profile of proteases (number, class, optimal conditions, and activity of the enzymes), and possible pathogenicity mechanism of the proteolytic enzymes (various protein substrate degradation, cytopathic effect on different cell lines). In some cases, it was attempted to correlate intracellular and extracellular protease profile with pathogenicity potential of strains. This review revealed that the protease profile of different strains of Acanthamoeba was extremely complex, therefore, further comprehensive studies with application of a combination of various methods may help to elucidate the role of the enzymes. [ABSTRACT FROM AUTHOR]

In recent decades, biosynthesis of metal and (or) metal oxide nanoparticles using microbes is accepted as one of the most sustainable, cost-effective, robust, and green processes as it does not encompass the usage of largely hazardous chemicals. Accordingly, numerous simple, inexpensive, and environmentally friendly approaches for the biosynthesis of silver nanoparticles (AgNPs) were reported using microbes avoiding conventional (chemical) methods. This comprehensive review detailed an advance made in recent years in the microbes-mediated biosynthesis of AgNPs and evaluation of their antimicrobial activities covering the literature from 2015-till date. It also aimed at elaborating the possible effect of the different phytochemicals, their concentrations, extraction temperature, extraction solvent, pH, reaction time, reaction temperature, and concentration of precursor on the shape, size, and stability of the synthesized AgNPs. In addition, while trying to understand the antimicrobial activities against targeted pathogenic microbes the probable mechanism of the interaction of produced AgNPs with the cell wall of targeted microbes that led to the cell's reputed and death have also been detailed. Lastly, this review detailed the shape and size-dependent antimicrobial activities of the microbes-mediated AgNPs and their enhanced antimicrobial activities by synergetic interaction with known commercially available antibiotic drugs.

Background: Expression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target. Methods: To support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated. Results: IHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term ‘ZP3-Cancer’, was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer. Conclusion: The cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies. [ABSTRACT FROM AUTHOR]

The intracellular protein corona has not been fully investigated in the field of nanotechnology-biology (nano-bio) interactions. To effectively understand intracellular protein corona formation and dynamics, we established a workflow to isolate the intracellular protein corona at different uptake times of two nanoparticles - magnetic hydroxyethyl starch nanoparticles (HES-NPs) and magnetic human serum albumin nanocapsules (HSA-NCs). We performed label-free quantitative LC-MS proteomics to analyze the composition of the intracellular protein corona and correlated our findings with results from conventional methods for intracellular trafficking of nanocarriers, such as flow cytometry, transmission electron microscopy (TEM), and confocal microscopy (cLSM). We determined the evolution of the intracellular protein corona. At different time stages the protein corona of the HES-NPs with a slower uptake changed, but there were fewer changes in that of the HSA-NCs with a more rapid uptake. We identified proteins that are involved in macropinocytosis (RAC1, ASAP2) as well as caveolin. This was confirmed by blocking experiments and by TEM studies. The investigated nanocarrier predominantly trafficked from early endosomes as determined by RAB5 identification in proteomics and in cLSM to late endosomes/lysosomes (RAB7, LAMP1, cathepsin K and HSP 90-beta) We further demonstrated differences between nanoparticles with slower and faster uptake kinetics and determined the associated proteome at different time points. Analysis of the intracellular protein corona provides us with effective data to examine the intracellular trafficking of nanocarriers used in efficient drug delivery and intracellular applications. Many research papers focus on the protein corona on nanoparticles formed in biological fluids, but there are hardly any articles dealing with proteins that come in contact with nanoparticles inside cells. The "intracellular protein corona" studied here is a far more complex and highly demanding field. Most nanocarriers are designed to be taken up into cells. Given this, we chose two different nanocarriers to reveal changes in the proteins in dendritic cells during contact at specific times. Further studies will allow us to examine molecular target proteins using these methods. Our research is a significant addition towards the goal of understanding and thus improving the efficacy of drug nanocarriers. [Display omitted] [ABSTRACT FROM AUTHOR]

Aim: To determine the effect of a novel antimicrobial peptide (AMP; OP145) and cell‐penetrating peptide (Octa‐arginine/R8) conjugate on the killing of intracellular Enterococcus faecalis, compared to OP145 and an antibiotic combination recommended for regenerative endodontic procedures. Methodology: The biocompatible concentrations of OP145 and OP145‐R8 were determined by assessing their cytotoxicity against human macrophages and red blood cells. Spatiotemporal internalization of the peptides into macrophages was investigated qualitatively and quantitatively by confocal laser scanning microscopy and flow cytometry respectively. Killing of extracellular and intracellular E. faecalis OG1RF by the peptides was determined by counting the colony‐forming units (CFU). Intracellular antibacterial activity of the peptides was compared to a double antibiotic combination. Confocal microscopy was used to confirm the intracellular bacterial eradication. Significant differences between the different test groups were analysed using one‐way analysis of variance. p <.05 was considered to be statistically significant. Results: Peptides at a concentration of 7.5 μmol/L were chosen for subsequent experiments based on the results of the alamarBlue™ cell viability assay and haemolytic assay. OP145‐R8 selectively internalized into lysosomal compartments and the cytosol of macrophages. Conjugation with R8 improved the internalization of OP145 into macrophages in a temporal manner (70.53% at 1 h to 77.13% at 2 h), while no temporal increase was observed for OP145 alone (60.53% at 1 h with no increase at 2 h). OP145‐R8 demonstrated significantly greater extracellular and intracellular antibacterial activity compared to OP145 at all investigated time‐points and concentrations (p <.05). OP145‐R8 at 7.5 μmol/L eradicated intracellular E. faecalis after 2 h (3.5 log reduction compared to the control; p <.05), while the antibiotics could not reduce more than 0.5 log CFU compared to the control (p >.05). Confocal microscopy showed complete absence of E. faecalis within the OP145‐R8 treated macrophages. Conclusions: The results of this study demonstrated that the conjugation of an AMP OP145 to a cell‐penetrating peptide R8 eradicated extracellular and intracellular E. faecalis OG1RF without toxic effects on the host cells. [ABSTRACT FROM AUTHOR]

Tumors and cysts with odontogenic origin represent a family of lesions with specific histo-genetic and clinical characteristics. Among them, ameloblastomas are common benign neoplasms, predominantly detected in the anatomic areas of the jaws and also in the mandible and maxilla. Although they are characterized by a slow and stable growing pattern, a subset of them shows a tendency for local tissue invasiveness and partially increased recurrence rates after surgical excision. Furthermore, heat shock proteins (HSPs) are potentially implicated in ameloblastoma onset and progression. HSPs regulate the folding and refolding of proteins and are induced in response to oxidative stress. They are crucial members of the chaperone intracellular system and are categorized based on their molecular weight (i.e., HSP27, HSP60, HSP70, HSP90). In the current review, we describe HSPs origin and function, focusing on their deregulation mechanisms and impact predominantly on ameloblastomas and also on inflammatory and developmental odontogenic cystic lesions. [ABSTRACT FROM AUTHOR]

The complement system, an arm of the innate immune system plays a critical role in both health and disease. The complement system is highly complex with dual possibilities, helping or hurting the host, depending on the location and local microenvironment. The traditionally known functions of complement include surveillance, pathogen recognition, immune complex trafficking, processing and pathogen elimination. The noncanonical functions of the complement system include their roles in development, differentiation, local homeostasis and other cellular functions. Complement proteins are present in both, the plasma and on the membranes. Complement activation occurs both extra‐ and intracellularly, which leads to considerable pleiotropy in their activity. In order to design more desirable and effective therapies, it is important to understand the different functions of complement, and its location‐based and tissue‐specific responses. This manuscript will provide a brief overview into the complex nature of the complement cascade, outlining some of their complement‐independent functions, their effects at different locale, and their implication in disease settings. [ABSTRACT FROM AUTHOR]

Methylorubrum extorquens DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derived methanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont M. extorquens DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (phaC), PHB depolymerase (phaZ1), and a transcription factor (phaR) showed altered PHB granule characteristics, as ΔphaC had a significantly low number of granules, ΔphaR had a significantly increased number of granules, and ΔphaZ1 had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the ΔphaC strain was sensitive to 10 mM HO· and 20 mM H2O2. The colonization of the host, Scots pine (Pinus sylvestris L.), by the deletion strains varied greatly. The deletion strain ΔphaR colonized the host mainly intercellularly, whereas the ΔphaZ1 strain was a slightly poorer colonizer than the control. The deletion strain ΔphaC lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by PhaC and PhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont M. extorquens DSM13060. Our current results show that the low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in M. extorquens DSM13060 is an important trait in the non-rhizobial endosymbiosis.

Nicotinamide adenine dinucleotide (NAD+), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD+ was primarily considered to be an intracellular molecule (iNAD+), however, its extracellular species (eNAD+) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD+ in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD+ concentrations. Initially, NAD+ levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD+ in plasma was found to be approximately 500 times lower than iNAD+ in peripheral blood mononuclear cells (0.253 ± 0.02 μM vs. 131.8 ± 27.4 μM, p = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD+ in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD+ (n = 33; r = −0.397; p = 0.102). Furthermore, eNAD+ was negatively correlated with increasing c-reactive protein (CRP, n = 33; r = −0.451; p = 0.020), while eNAD+ was positively correlated with increasing hemoglobin (n = 33; r = 0.482; p = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0–120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 – 120 s) had no statistically significant effect on eNAD+ (p > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (< 4 min) resulted in a significant decrease in eNAD+ levels (p

We report innovative scalable, vertical, ultra-sharp nanowire arrays that are individually addressable to enable long-term, native recordings of intracellular potentials. Stable amplitudes of intracellular potentials from 3D tissue-like networks of neurons and cardiomyocytes are obtained. Individual electrical addressability is necessary for high-fidelity intracellular electrophysiological recordings. This study paves the way toward predictive, high-throughput, and low-cost electrophysiological drug screening platforms.

This study presents a biosynthetic route for TiO2-doped ZnO (Ti/ZnO) using the primary metabolite component of the Aspergillus niger fungi as a source of the capping agent. The effect of biosynthetic variables based on the extracellular (Ti/ZnO) and intracellular (Ti/ZnO) enzymatic cycles of Aspergillus niger during the 3–4 days growth period was analyzed. The morphological differences between the extracellular and intracellular Ti/ZnO produced were characterized using Fourier Transform Infrared (FTIR) spectroscopy, Ultraviolet-Visible Spectrometry (UV–Vis), X-ray Diffraction (XRD), and Scanning Electron Microscope equipped with Energy Dispersive X-ray spectroscopy (SEM-EDX). Analyzing Ti/ZnO with different biosynthetic variables exhibited the same crystal structure, namely hexagonal Wurtzite, but with different morphology and crystal sizes of 27.42–28.99 nm. Furthermore, Ti/ZnO has a pattern of a rod with a length and diameter of 287–455 nm and 126 - 156 nm, respectively. In vitro antimicrobial activity testing against Staphylococcus epidermidis bacterial cells was performed under exposure to ultraviolet light with the wavelength of λ = 385 nm. The results indicated that the increase in bacterial activity was influenced by the effect of biosynthetic variables based on differences in morphology, size, the shape of Ti/ZnO, enzymatic cycle, and growth time of Aspergillus niger.

BackgroundExpression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target.MethodsTo support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated.ResultsIHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term ‘ZP3-Cancer’, was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer.ConclusionThe cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies.

Although considerable attention has been paid to the role of extracellular galectins in modulating, positively or negatively, tumor growth and metastasis, we have witnessed a growing interest in the role of intracellular galectins in response to their environment. This is not surprising as many galectins preferentially exist in cytosolic and nuclear compartments, which is consistent with the fact that they are exported outside the cells via a yet undefined nonclassical mechanism. This review summarizes our most recent knowledge of their intracellular functions in cancer cells and provides some directions for future strategies to inhibit their role in cancer progression. [ABSTRACT FROM AUTHOR]

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians. [ABSTRACT FROM AUTHOR]

Although electrophysiologists have been recording intracellular neural activity routinely ever since the ground‐breaking work of Hodgkin and Huxley, and extracellular multichannel electrodes have also been used frequently and extensively, a practical experimental method to track changes in membrane potential along a complete single neuron is still lacking. Instead of obtaining multiple intracellular measurements on the same neuron, we propose an alternative method by combining single‐channel somatic patch‐clamp and multichannel extracellular potential recordings. In this work, we show that it is possible to reconstruct the complete spatiotemporal distribution of the membrane potential of a single neuron with the spatial resolution of an extracellular probe during action potential generation. Moreover, the reconstruction of the membrane potential allows us to distinguish between the two major but previously hidden components of the current source density (CSD) distribution: the resistive and the capacitive currents. This distinction provides a clue to the clear interpretation of the CSD analysis, because the resistive component corresponds to transmembrane ionic currents (all the synaptic, voltage‐sensitive and passive currents), whereas capacitive currents are considered to be the main contributors of counter‐currents. We validate our model‐based reconstruction approach on simulations and demonstrate its application to experimental data obtained in vitro via paired extracellular and intracellular recordings from a single pyramidal cell of the rat hippocampus. In perspective, the estimation of the spatial distribution of resistive membrane currents makes it possible to distiguish between active and passive sinks and sources of the CSD map and the localization of the synaptic input currents, which make the neuron fire. Key points: A new computational method is introduced to calculate the unbiased current source density distribution on a single neuron with known morphology.The relationship between extracellular and intracellular electric potential is determined via mathematical formalism, and a new reconstruction method is applied to reveal the full spatiotemporal distribution of the membrane potential and the resistive and capacitive current components.The new reconstruction method was validated on simulations.Simultaneous and colocalized whole‐cell patch‐clamp and multichannel silicon probe recordings were performed from the same pyramidal neuron in the rat hippocampal CA1 region, in vitro.The method was applied in experimental measurements and returned precise and distinctive characteristics of various intracellular phenomena, such as action potential generation, signal back‐propagation and the initial dendritic depolarization preceding the somatic action potential. [ABSTRACT FROM AUTHOR]

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a “pre-antibiotic” type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method. [ABSTRACT FROM AUTHOR]

Pathogen environmental stability is an often-neglected research priority for pathogens that are known to be vector-transmitted. Bartonella henselae, the etiologic agent of Cat Scratch Disease, has become a "pathogen of interest" in several serious human illnesses, which include neoplastic, cardiovascular, neurocognitive, and rheumatologic conditions. Survival in the flea gut and feces as well as the association with a biofilm in culture-negative endocarditis provides insight into this organism's ability to adjust to environmental extremes. The detection of B. henselae DNA in blood and tissues from marine mammals also raises questions about environmental stability and modes of pathogen transmission. We investigated the ability of B. henselae to survive in fluid matrices chosen to mimic potential environmental sources of infective materials. Feline whole blood, serum and urine, bovine milk, and physiologic saline inoculated with a laboratory strain of B. henselae San Antonio 2 were subsequently evaluated by culture and qPCR at specified time intervals. Bacterial viability was also assessed following desiccation and reconstitution of each inoculated fluid matrix. Bartonella henselae SA2 was cultured from feline urine up to 24 h after inoculation, and from blood, serum, cow's milk, and physiologic saline for up to 7 days after inoculation. Of potential medical importance, bacteria were cultured following air-desiccation of all fluid inoculates. The viability and stability of Bartonella within biological and non-biological fluids in the environment may represent a previously unrecognized source of infection for animals and human beings. [ABSTRACT FROM AUTHOR]

Although considerable attention has been paid to the role of extracellular galectins in modulating, positively or negatively, tumor growth and metastasis, we have witnessed a growing interest in the role of intracellular galectins in response to their environment. This is not surprising as many galectins preferentially exist in cytosolic and nuclear compartments, which is consistent with the fact that they are exported outside the cells via a yet undefined non-classical mechanism. This review summarizes our most recent knowledge of their intracellular functions in cancer cells and provides some directions for future strategies to inhibit their role in cancer progression.

In humans, a total of 12 galectins have been identified. Their intracellular and extracellular biological functions are explored and discussed in this review. These galectins play important roles in controlling immune responses within the tumour microenvironment (TME) and the infiltration of immune cells, including different subsets of T cells, macrophages, and neutrophils, to fight against cancer cells. However, these infiltrating cells also have repair roles and are hijacked by cancer cells for pro-tumorigenic activities. Upon a better understanding of the immunomodulating functions of galectin-3 and -9, their inhibitors, namely, GB1211 and LYT-200, have been selected as candidates for clinical trials. The use of these galectin inhibitors as combined treatments with current immune checkpoint inhibitors (ICIs) is also undergoing clinical trial investigations. Through their network of binding partners, inhibition of galectin have broad downstream effects acting on CD8+ cytotoxic T cells, regulatory T cells (Tregs), Natural Killer (NK) cells, and macrophages as well as playing pro-inflammatory roles, inhibiting T-cell exhaustion to support the fight against cancer cells. Other galectin members are also included in this review to provide insight into potential candidates for future treatment(s). The pitfalls and limitations of using galectins and their inhibitors are also discussed to cognise their clinical application. [ABSTRACT FROM AUTHOR]

The use of self‐assembly for building complex functional structures is a current topic of interest in supramolecular chemistry. In this context, the use of biomolecule‐based building blocks has paved the way for the development of intracellular assemblies. Currently, the potential functionality of such assemblies in biomedical applications is being disclosed. On the other hand, the use of inorganic (metal‐based) building blocks is still in its infancy. The construction of inorganic self‐assemblies in‐bio is particularly challenging and demands great efforts to reach applications. However, the plethora of thinkable advantages related to the use of inorganic self‐assembly in living cells must fuel new discoveries in this area. This Concept reviews the current advances, perspectives, and challenges in inorganic self‐assembly in living systems. [ABSTRACT FROM AUTHOR]

Background: Insight into the pathomechanism of atopic diseases demonstrated a pivotal role of the cytokines interleukin‐4 (IL‐4) and IL‐13, which has spurred the development of tailored therapeutics targeting their common IL‐4 receptor (IL‐4R). However, several aspects of the IL‐4R system remain ill‐defined in humans. Methods: We used multicolor spectral flow cytometry to characterize IL‐4R subunit expression in 28 human immune cell subsets on protein and mRNA levels and assessed their subcellular distribution by applying a specifically adapted protocol that avoided influence of fixation and permeabilization on fluorochrome and antibody performance. In patients, we investigated possible changes in IL‐4Rα distribution before and during treatment with dupilumab, a monoclonal antibody‐targeting IL‐4Rα. Results: Whereas all immune cell subsets investigated expressed IL‐4Rα and common γ chain protein and mRNA, expression of IL‐13Rα1 was restricted to myeloid and B cells. Interestingly, some cells contained considerably more intracellular IL‐4R protein than on their surface. Naive B cells were found to carry the highest levels of IL‐4Rα distributed evenly between surface and intracellular space, whereas IL‐4Rα was found predominantly in intracellular pools in neutrophils. In patients with atopic diseases treated with dupilumab, we observed that engagement of IL‐4Rα by dupilumab resulted in internalization of the antibody and decreased total IL‐4Rα expression. Notably, even after months of treatment not all intracellular IL‐4Rα molecules were occupied by dupilumab, indicating the presence of a "dormant" intracellular IL‐4Rα pool that could be mobilized upon certain extrinsic or intrinsic cues. Conclusion: Collectively, our findings suggest that distinct human immune cell subsets contain surface and intracellular IL‐4R pools, which are differently affected by targeted biologic treatment. [ABSTRACT FROM AUTHOR]

In recent years, Mycobacterium abscessus has appeared as an emerging pathogen, with an increasing number of disease cases reported worldwide that mainly occur among patients with chronic lung diseases or impaired immune systems. The treatment of this pathogen represents a challenge due to the multi-drug-resistant nature of this species and its ability to evade most therapeutic approaches. However, although predisposing host factors for disease are well known, intrinsic pathogenicity mechanisms of this mycobacterium are still not elucidated. Like other mycobacteria, intracellular invasiveness and survival inside different cell lines are pathogenic factors related to the ability of M. abscessus to establish infection. Some of the molecular factors involved in this process are well-known and are present in the mycobacterial cell wall, such as trehalose-dimycolate and glycopeptidolipids. The ability to form biofilms is another pathogenic factor that is essential for the development of chronic disease and for promoting mycobacterial survival against the host immune system or different antibacterial treatments. This capability also seems to be related to glycopeptidolipids and other lipid molecules, and some studies have shown an intrinsic relationship between both pathogenic mechanisms. Antimicrobial resistance is also considered a mechanism of pathogenicity because it allows the mycobacterium to resist antimicrobial therapies and represents an advantage in polymicrobial biofilms. The recent description of hyperpathogenic strains with the potential interhuman transmission makes it necessary to increase our knowledge of pathogenic mechanisms of this species to design better therapeutic approaches to the management of these infections. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

Harmful cyanobacterial blooms are increasing in frequency and severity, which makes their toxic secondary metabolites of microcystins (MCs) have been widely studied, especially in their distribution and influence factors in different habitats. However, the distribution of MCs on the early growth stage of harmful cyanobacteria and its influence factors and risks are still largely unknown. Thus, in the present study, two reservoirs (Lutian Reservoir and Lake Haitang) with different trophic status in China have been studied weekly from March to May in 2018, when the cyanobacteria communities were just in the early growth stage, to investigate the variation of MCs concentration and the relationships between MCs and environmental parameters. During the investigation, Lutian Reservoir and Lake Haitang were found to be mesotrophic and light eutrophic, respectively. In Lutian Reservoir, the concentration of EMCs (extracellular MCs) was obviously higher than that of IMCs (intracellular MCs) with a mean value of 0.323 and 0.264 μg/L, respectively. Meanwhile, the concentration of EMCs also fluctuated more sharply than that of IMCs. Congeners of IMC-YR and EMC-LR were respectively dominant in total concentrations of IMCs and EMCs. Unsurprisingly, in Lake Haitang, the concentrations of IMC and EMC were both significantly higher than that in Lutian Reservoir with a mean concentration of 0.482 and 0.472 μg/L, respectively. Differently, the concentration of MC-YR was dominant in both IMCs and EMCs, followed by MC-LR. In correlation analysis, the IMCs were significantly and positively correlated with the density and biomass of phytoplankton phyla and potential MCs-producing cyanobacteria and the parameters of water temperature (WT), nutrients, and organic matters. Similar results were also observed for EMCs. While the different variations of MCs in the two reservoirs might be primarily caused by the differences in WT, nutrients (especially phosphorus), organic matters, and the composition of MCs-producing cyanobacteria. In addition, the coexistence of the dominant species of Pseudoanabaena sp., which can produce a taste-and-odor compound of 2-methylisoborneol (2-MIB), might have a significant impact on the concentration and toxicity of MCs. Our results suggested that the risks posed by MCs at the early growth stage of cyanobacteria should also deserve our attention, especially in mesotrophic water bodies. [ABSTRACT FROM AUTHOR]

Plant polysaccharides are components of plant cell walls and/or store energy. However, this oversimplified classification neglects the fact that some cell wall polysaccharides and glycoproteins can localize outside the relatively sharp boundaries of the apoplastic moiety, where they adopt functions not directly related to the cell wall. Such polysaccharide multifunctionality (or 'moonlighting') is overlooked in current research, and in most cases the underlying mechanisms that give rise to unconventional ex muro trafficking, targeting, and functions of polysaccharides and glycoproteins remain elusive. This review highlights major examples of the extramural occurrence of various glycan cell wall components, discusses the possible significance and implications of these phenomena for plant physiology, and lists exciting open questions to be addressed by future research. A large amount of evidence has accumulated over the last decades that polysaccharides and other cell wall components such as glycoproteins can be targeted ex muro (outside the conventionally defined cell wall space). The trafficking and functions of extramural cell wall components located either intracellularly or ex planta are mostly speculative or require more attention from plant researchers. We propose that (i) the functional plasticity and extramural distribution of cell wall-related polysaccharides are based on so far overlooked or uncharacterized transport systems that bypass and/or complement conventional polysaccharide secretion routes, and that (ii) compositional variations in a cell wall polysaccharide could determine a new role, depending on its final destination. [ABSTRACT FROM AUTHOR]

Non-tuberculous mycobacteria are widely distributed in environments and are capable of infecting humans, particularly those with a compromised immune system. The most prevalent species that cause nontuberculous mycobacterial lung diseases are slow-growing bacteria from the Mycobacterium avium complex (MAC), mainly M. avium or M. intracellulare. The key treatment of MAC infections includes macrolides, ethambutol, and rifampicin; however, the therapy outcomes are unsatisfactory. Phenotypic drug susceptibility testing is a conditional recommendation prior to treatment, and critical concentrations for clarithromycin, amikacin, moxifloxacin, and linezolid have been established. In this review, data from studies on the determination of MIC of clinical isolates using the broth microdilution method were summarized. A significant variation in the MIC distributions from different studies was found. The main reasons could impact the findings: insufficient reproducibility of the phenotypic testing and variation in species lineages identified in different laboratories, which could have various intrinsic susceptibility to drugs. For most of the drugs analyzed, the MICs are too high, which could undermine the treatment efficiency. Further improvement of treatment outcomes demands the validation of microbiological resistance criteria together with the identification of molecular mechanisms of resistance. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium infects eukaryotic cells residing within membrane‐bound phagosomes. In this compartment, the pathogen replaces the morphogenetic penicillin‐binding proteins 2 and 3 (PBP2/PBP3) with PBP2SAL/PBP3SAL, two proteins absent in Escherichia coli. The basis for this switch is unknown. Here, we show that PBP3 protein levels drop drastically when S. Typhimurium senses acidity, high osmolarity and nutrient scarcity, cues that activate virulence functions required for intra‐phagosomal survival and proliferation. The protease Prc and the transcriptional regulator OmpR contribute to lower PBP3 levels whereas OmpR stimulates PBP2SAL/PBP3SAL production. Surprisingly, despite being essential for division in E. coli, PBP3 levels also drop in non‐pathogenic and pathogenic E. coli exposed to phagosome cues. Such exposure alters E. coli morphology resulting in very long bent and twisted filaments indicative of failure in the cell division and elongation machineries. None of these aberrant shapes are detected in S. Typhimurium. Expression of PBP3SAL restores cell division in E. coli exposed to phagosome cues although the cells retain elongation defects in the longitudinal axis. By switching the morphogenetic program, OmpR and Prc allow S. Typhimurium to properly divide and elongate inside acidic phagosomes maintaining its cellular dimensions and the rod shape. [ABSTRACT FROM AUTHOR]

• PK/PD study of current regimens during early treatment of primary HIV infection. • There were no significant differences between treatment arms. • Differences in ARV concentrations in plasma, PBMCs and LNs were described. • Higher HIV DNA decay in patients with low Fiebig stages. • TFV–DP in PBMCs was correlated with HIV DNA decay in patients with higher Fiebig stages. Despite its effectiveness, combination antiretroviral treatment (cART) has a limited effect on HIV DNA reservoir, which establishes early during primary HIV infection (PHI) and is maintained by latency, homeostatic T-cells proliferation, and residual replication. This limited effect can be associated with low drug exposure in lymphoid tissues and/or suboptimal adherence to antiretroviral drugs (ARVs). The aim of this study was to assess ARV concentrations in plasma, peripheral blood mononuclear cells (PBMCs) and lymph nodes (LNs), and their association to HIV RNA and HIV DNA decay during PHI. Participants were randomised to receive standard doses of darunavir/cobicistat (Arm I), dolutegravir (Arm II) or both (Arm III), with a backbone of tenofovir alafenamide and emtricitabine. Total HIV DNA was measured using digital-droplet PCR in PBMCs at baseline, 12 and 48 weeks. Drug concentrations in plasma and PBMCs were determined at 2, 12 and 48 weeks (LNs at 12 weeks) by UHPLC-MS/MS. Seventy-two participants were enrolled, mostly male (n=68), with a median age of 34 years and variable Fiebig stages (V–VI 57.7%, I–II 23.9%, and III–IV 18.3%). Twenty-six patients were assigned to Arm I, 27 to Arm II and 19 to Arm III. After 48 weeks, most patients had undetectable viremia, with minor differences in HIV RNA decay between arms. Patients with Fiebig I–II showed faster HIV RNA and HIV DNA decay. Intracellular tissue penetration was high for nucleoside analogues and low-moderate for darunavir and dolutegravir. Only tenofovir diphosphate concentrations in PBMCs showed correlation with HIV DNA decay. Overall, these results indicate that the timing of treatment initiation and intracellular tenofovir penetration are primary and secondary factors, respectively, affecting HIV reservoir. [Display omitted] [ABSTRACT FROM AUTHOR]

Pathogen environmental stability is an often-neglected research priority for pathogens that are known to be vector-transmitted. Bartonella henselae, the etiologic agent of Cat Scratch Disease, has become a “pathogen of interest” in several serious human illnesses, which include neoplastic, cardiovascular, neurocognitive, and rheumatologic conditions. Survival in the flea gut and feces as well as the association with a biofilm in culture-negative endocarditis provides insight into this organism’s ability to adjust to environmental extremes. The detection of B. henselae DNA in blood and tissues from marine mammals also raises questions about environmental stability and modes of pathogen transmission. We investigated the ability of B. henselae to survive in fluid matrices chosen to mimic potential environmental sources of infective materials. Feline whole blood, serum and urine, bovine milk, and physiologic saline inoculated with a laboratory strain of B. henselae San Antonio 2 were subsequently evaluated by culture and qPCR at specified time intervals. Bacterial viability was also assessed following desiccation and reconstitution of each inoculated fluid matrix. Bartonella henselae SA2 was cultured from feline urine up to 24 h after inoculation, and from blood, serum, cow’s milk, and physiologic saline for up to 7 days after inoculation. Of potential medical importance, bacteria were cultured following air-desiccation of all fluid inoculates. The viability and stability of Bartonella within biological and non-biological fluids in the environment may represent a previously unrecognized source of infection for animals and human beings.

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.