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Background: The second phase of the U.S. President's Emergency Plan for AIDS Relief (PEPFAR) transitioned from scaling up HIV prevention and treatment to promoting sustainability and capacity building for programs monitoring performance and evaluating key program indicators. We assessed the success of a monitoring and evaluation (M&E) curriculum designed to build capacity in three PEPFAR-supported countries., Methods: We customized M&E trainings based on country-specific epidemic control priorities in Ethiopia, Guatemala, and Cameroon. The M&E curriculum included five modules and three evaluation activities to assess impact: (i) in-person pre-post confidence assessment surveys (CAS), (ii) in-person pre-post knowledge tests (PPKT), and (iii) electronic 6-12 months post-training translating knowledge into practice (TKP) surveys. Pre- and post-training results were compared within and across countries and triangulation with the qualitative data evaluated overall success., Results: Among 188 participants attending M&E trainings, 154 (82 %) responded to CAS and 165 (88 %) participants from Ethiopia and Cameroon completed PPKT. Overall CAS scores between pre- and post-test improved [Score mean difference:1.5-1.9]. PPKT indicated statistically significant knowledge gained. One out of five TKP respondents provided direct application examples from the M&E training., Conclusion: While feedback was predominantly positive overall, revisions were recommended for three of the five modules. Developing a customizable and adaptable M&E curriculum may sustain countries' ability to monitor their progress towards epidemic control., (Copyright © 2024. Published by Elsevier Ltd.)

The Solanaceae family and the Withania genus specifically are rich sources of medicinal plants. Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS/MS) revealed a predominance of withanolides from an organic extract of Withania obtusifolia. A constructed molecular network uncovered the presence of potentially novel withanolides. A series of withanolides were then isolated and structurally characterized from the extract including two new withanolides (withafolia A and withafolia B) and seven previously reported metabolites. Of the isolated compounds, cytotoxicity of withanolide J, physaperuvin G, and a commercial STAT3 inhibitor (S3I-201) were assessed against a human leukemia HL-60 cell line resulting in IC 50 values of 26, 29, and 120 μM, respectively. In silico molecular docking simulations indicate that withanolide J and physaperuvin G can bind as an inhibitor in the active site of STAT3 with docking scores comparable to the selective STAT3 inhibitor, S3I-201., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paul W Pare reports financial support and equipment, drugs, or supplies were provided by Texas Tech University. If there are other authors, they 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. Published by Elsevier B.V.)

Background and Objectives: Systematic reviews form the basis of evidence-based medicine, but are expensive and time-consuming to produce. To address this burden, we have developed a literature identification system (Pythia) that combines the query formulation and citation screening steps., Methods: Pythia incorporates a set of natural-language questions with machine-learning algorithms to rank all PubMed citations based on relevance, returning the 100 top-ranked citations for human screening. The tagged citations are iteratively exploited by Pythia to refine the search and re-rank the citations., Results: Across seven systematic reviews, the ability of Pythia to identify the relevant citations (sensitivity) ranged from 0.09 to 0.58. The number of abstracts reviewed per relevant abstract number needed to read (NNR) was lower than in the manually screened project in four reviews, higher in two, and had mixed results in one. The reviews that had greater overall sensitivity retrieved more relevant citations in early batches, but retrieval was generally unaffected by other aspects, such as study design, study size, and specific key question., Conclusion: Due to its low sensitivity, Pythia is not ready for widespread use. Future research should explore ways to encode domain knowledge in query formulation to better enrich the questions used in the search., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Blood is a routinely tested biological fluid for diagnosis and monitoring of diseases as many diseases would trigger a change in white blood cell count. Thus, several methods have been established to isolate or enrich white blood cells from patient blood samples for such analyses. One method of preparing an enriched white blood cell sample is through the selective lysis of red blood cells by hypotonic shock and restoration of osmolarity to maintain viability of target white blood cells. An inherent problem with this approach is the loss of target cells during sample handling. We report a two-stage separation system that can perform lysis and restoration of osmolarity of blood on-chip and direct the resultant sample to the second step of the analysis. Hence, there is no loss of sample. The post-lysis makeup features a protein-rich buffer to help stabilize cells. As proof of concept, we spiked HL-60 cells into a whole blood and a pre-lysed blood sample and compared capture metrics of each method using a downstream affinity separation. The capture efficiency of the whole blood sample ranged between 40 and 80% using <7 μL of sample compared to 10-52% from 60 μL of pre-lysed blood required for similar analysis. In addition, both pre-lysed and whole blood samples showed no significant difference in purity and viability. This two-stage separation system has demonstrated the capacity to replace centrifugation and wash steps required for the preparation of lysed blood, for white blood cell analyses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cancer cell detection in liquid biopsies has been a widely studied application in many microfluidic devices. The use of a common antibody, such as the epithelial cell adhesion molecule (Anti-EpCAM) or other specific antibodies, has facilitated the detection and study of many cancers. However, the use of such antibodies requires a priori knowledge of the cancer source, and many cancer subtypes are missed in screening applications. There remains a need to study a wider range of cancers that maintain the streamlined antibody approach in cell affinity separations. The Human transferrin receptor (CD71) has recently been demonstrated as a cancer cell affinity target in blood samples. CD71 expression in blood cells is low, whereas proliferating cancer cells have a higher expression of the surface protein. CD71 expression is variable with cell cycle, which can impact cell separations. In this work, we investigated the effects of cell cycle and CD71 expression on cell capture metrics. Six cancer cell lines were isolated from blood via CD71 affinity capture, with a capture efficiency and purity that varied with CD71 expression. Despite variation in CD71 expression, the affinity was sufficient to isolate cancer cells spiked into blood; under optimal conditions, CD71-based capture resulted in capture purity >80%. We conclude that CD71 affinity separations show potential as a biomarker for cancer studies without sacrificing sensitivity and selectivity, and that cancer cells can be isolated from liquid biopsies over a range of expression of the target protein., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sepsis is a life-threatening disease that affects millions of people every year. Rapid detection of sepsis assists clinicians to initiate timely antibiotic therapy and to reduce mortality. At the same time, accurate point-of-care detection is needed to reduce unnecessary use of antibiotics. One of the principal challenges in sepsis diagnosis is that many sepsis cases do not result in positive blood cultures. These so-called culture-negative cases present a significant health threat. In this work, we present a microfluidic cells separation system for the detection of sepsis in both culture-positive and culture-negative cases. Leukocytes were captured in several affinity separation zones of a microchip based on CD64, CD69, and CD25 expression. To validate this assay 40 septic patients and 10 healthy volunteers were enrolled in this study. Septic patients were divided into culture-positive (n = 12) and culture-negative cases (n = 21). CD64  +  cell capture demonstrated excellent accuracy for sepsis detection with an area under the receiver operating characteristic curves (AUC) of 0.962. A combined panel of CD64  +  and CD69  +  cell counts was constructed, and the new panel outperformed each of these two biomarkers alone with the AUC of 0.978. Our affinity microfluidic devices were validated by conventional flow cytometry analysis. Results showed that the cell capture number of specific affinity region increased along with the increase of its corresponding antigen expression. This clinical validation confirms that CD64 and CD69 cell separations are a powerful sepsis assay with the potential for point-of-care analysis in culture-positive and culture-negative cases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sepsis is a highly prevalent syndrome in the United States. The use of cell surface markers, as an effective tool to diagnosis sepsis, has been widely investigated. However, the study of the combination of multiple biomarkers to achieve higher diagnosis accuracy is rare. This study, the panel combined with CD25, CD64, and CD69 was constructed and better diagnosis ability was observed. Septic patients (n = 40), with the mean age of 61 ± 14, were enrolled in this study, along with healthy volunteers (n = 10), included as a control group. All blood samples were measured by flow cytometry based on different subtypes of leukocytes, including neutrophils, monocytes, and lymphocytes. Antigen expression and the antigen positive cell population were reported separately based on cell types. CD64 was the best biomarker in predicting sepsis. The area under Receiver Operating Characteristic (ROC) curve (AUC) was 0.928 and 0.934 for neutrophil CD64 expression and CD64 + neutrophil population, respectively, indicating an excellent diagnosis ability for sepsis. A significant increase was also observed in the populations of CD25 + lymphocytes and CD69 + lymphocytes (p = 0.02 and 0.042, respectively; 95% confidence interval). A panel of combined CD25, CD64, and CD69 was constructed. The parameters of neutrophil CD64 expression, CD64 + neutrophil population, CD25 + lymphocyte population, and CD69 + lymphocyte population were included. The AUC of the ROC curve for this new constructed panel was 0.978. This result indicated that the combination of CD25, CD64, and CD69 outperformed each one of the single parameters in predicting sepsis alone., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This cross sectional study aimed to compare the differences in psychopathology of Greek homicide and homicide attempters, patients with schizophrenia, with non violent individuals, suffering from schizophrenia. The study compared three Groups of 220 men, diagnosed with schizophrenia: (a) Group Α (Schizophrenia - No violence, (b) Group Β (Schizophrenia - with violence or violent crime), (c) Group C (Schizophrenia - not guilty by reason of insanity - violent crime). Several psychometric tools were used, such as M.I.N·I (Mini-International Neuropsychiatric Interview), PANSS scale (Positive and Negative Symptoms Scale). Most subjects suffered from paranoid schizophrenia. On factors such as demographic characteristics (i.e. current occupational status, living status), statistically significant findings were shown for Groups B and C vs Group A. Predisposing psychosocial factors, such as family conflicts and aggressiveness against family, were found to be statistically significant in differentiating violent versus nonviolent individuals with psychosis. They differed significantly in factors like history of juvenile delinquency, but also in the type of aggressiveness in general. These differences were confirmed on PANSS scale. In conclusion, the longer the history of aggressiveness is presented, the greater the chances are of individuals falling into Group C and it is possible to spend several years from the onset of the disease until the moment of crime., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Microfluidic spatial and temporal gradient generators have played an important role in many biological assays such as in the analysis of wound healing, inflammation, and cancer metastasis. Chemical gradient systems can also be applied to other fields such as drug design, chemical synthesis, chemotaxis, etc. Microfluidic systems are particularly amenable to gradient formation, as the length scales used in chips enable fluid processes that cannot be conducted in bulk scale. In this review we discuss new microfluidic devices for gradient generation and applications of those systems in cell analysis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work, we demonstrate a two-layer microfluidic system capable of spatially selective delivery of drugs and other reagents under low shear stress. Loading occurs by hydrodynamically focusing a reagent stream over a particular region of the cell culture. The system consisted of a cell culture chamber and fluid flow channel, which were located in different layers to reduce shear stress on cells. Cells in the center of the culture chamber were exposed to parallel streams of laminar flow, which allowed fast changes to be made to the cellular environment. The shear force was reduced to 2.7 dyn cm(-2) in the two-layer device (vs. 6.0 dyn cm(-2) in a one-layer device). Cells in the side of the culture chamber were exposed to the side streams of buffer; the shear force was further reduced to a greater extent since the sides of the culture chamber were separated from the main fluid path. The channel shape and flow rate of the multiple streams were optimized for spatially controlled reagent delivery. The boundaries between streams were well controlled at a flow rate of 0.1 mL h(-1), which was optimized for all streams. We demonstrated multi-reagent delivery to different regions of the same culture well, as well as selective treatment of cancer cells with a built in control group in the same well. In the case of apoptosis induction using staurosporine, 10% of cells remained viable after 24 h of exposure. Cells in the same chamber, but not exposed to staurosporine, had a viability of 90%. This chip allows dynamic observation of cellular behavior immediately after drug delivery, as well as long-term drug treatment with the benefit of large cell numbers, device simplicity, and low shear stress., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this work, differential mobility cytometry (DMC) was used to monitor cell separation based on aptamer recognition for target cells. In this device, open-tubular capillaries coated with Sgc8 aptamers were used as affinity chromatography columns for separation. After cells were injected into the columns, oscillating flow was generated to allow for long-term cell adhesion studies. This process was monitored by optical microscopy, and differential imaging was used to analyze the cells as they adhered to the affinity surface. We investigated the capture time, capture efficiency, purity of target and control cells, as well as the reusability of the affinity columns. Capture time for both CCRF-CEM cells and Jurkat T cells was 0.4+/-0.2 s, which demonstrated the high separation affinity between aptamers and target cells. The capture efficiency for CCRF-CEM cells was 95% and purity was 99% in a cell mixture. With the advantage of both high cell capture efficiency and purity, DMC combined with aptamer-based separation emerges as a powerful tool for rare cell enrichment. In addition, aptamer-based DMC channels were found to be more robust than antibody based channels with respect to reuse of the separation device., (Copyright 2010 Elsevier B.V. All rights reserved.)

The ability to quickly measure flow parameters in microfluidic devices is critical for micro total analysis system (microTAS) applications. Macrofluidic methods to assess flow suffer from limitations that have made conventional methods unsuitable for the flow behavior profiling. Single molecule fluorescence correlation spectroscopy (FCS) has been employed in our study to characterize the fluidic vortex generating at a T-shape junction of microscale channels. Due to its high spatial and temporal resolution, the corresponding magnitudes relative to different flow rates in the main channel can be quantitatively differentiated using flow time (tau(F)) measurements of dye molecules traversing the detection volume in buffer solution. Despite the parabolic flow in the channel upstream, a heterogeneous distribution of flow has been detected across the channel intersection. In addition, our current observations also confirmed the aspect of vortex-shaped flow in low-shear design that was developed previously for cell culture. This approach not only overcomes many technical barriers for examining hydrodynamic vortices and movements in miniature structures without physically integrating any probes, but it is also especially useful for the hydrodynamic studies in polymer-glass based micro-reactor and -mixer.

In glass/poly(dimethylsiloxane) (PDMS) hybrid microfluidic chips, two different fabrication approaches are used: photolithographic or solid ink molds, or cast-and-peel methods. In the latter, a thin slab of PDMS is laid down and fluid channels are cut manually or by machine. The cast-and-peel approach has been used successfully for low-shear culture devices, among other applications. The main drawback, not reported to date, of cast-and-peel methods is that removal of PDMS (exposing the glass substrate) results in nanoscopic domains of PDMS still attached to the surface. This residual PDMS is not observable by eye, but affects the hydrophobicity of the device. Using contact angle measurement, atomic force and fluorescence microscopy, the changes in glass surfaces from the cast-and-peel technique were elucidated. This study demonstrates the enhanced protein (NeutrAvidin) adsorption on PDMS treated glass surfaces, and the potential influence of altered glass properties on microfluidic applications has been discussed as well.

A simple device for the separation of cells by phenotype is described. Cells are separated/isolated using capture antibodies on a glass chip. Unlike other "sandwich" type assays, the readout is performed without labels using transmission microscopy, simplifying cell enumeration. T and B lymphocytes from cell culture or whole blood were separated using antibodies for the CD4, CD19, and CD71 antigens. The separation slides were found to reproducibly bind cells by antigen expression, allowing for accurate enumeration of mixed cell samples. Inter- and intra-device variability was evaluated, and the issue of nonspecific binding is addressed. We envision that this type of cell separation technique could be used in remote settings, as sample preparation is minimal and the analysis time is rapid (20 min from sample acquisition to final readout).

The ability to generate a sample of cells of a given phenotype is a prerequisite for many cellular assays. In response to this growing need, numerous methods for cell separation have been developed in recent years. This Review covers recent progress in the field of cell separations and cell chromatography. Cell separation principles-such as size and affinity capture-are discussed, as well as conventional methods such as fluorescence-activated cell sorting and magnetic sorting. Planar flow cell arrays, dielectrophoresis, field-flow methods, and column separation devices are reviewed, as well as applications of these methods to medicine and biotechnology. Cell attachment and adhesion strategies and a comparison of techniques are also presented.

A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.

Recent advances in instrument design have allowed researchers to use smaller, more efficient components in designing Raman spectrometers. Advances in instrumentation have increased the performance of Raman instruments and increased their usage in bioanalysis. This paper reviews recent improvements in instrument design and discusses several applications of Raman spectroscopy to diagnosis in biology, chemistry and medicine.