The role of advocacy in sustaining male contraceptive research and development.

Novel male contraceptives have been in development for well over half a century, and despite a robust predicted global market for new methods, funding for research and development has been extremely limited. While the pharmaceutical industry previously supported male contraceptive research and development, industry partners are only spectators in the current space, awaiting a product that has been de-risked by the public sector before re-entering the field. Current male contraceptive development efforts are thus primarily funded by nonprofit, non-governmental, and government agencies who also act as the primary advocates for the field. Specific organizations include the International Consortium on Male Contraception, the Population Council, the Male Contraceptive Initiative, the World Health Organization, and the US National Institutes of Health. The funding provided by these public agencies, alongside their social and policy-based advocacy efforts such as market research, public education, and calls to action have kept the male contraceptive product development space afloat, resulting in a pipeline of potential products advancing towards market approval. However, as these products mature into more expensive clinical stages of development, they continue to face significant funding challenges, which many programs may not overcome. To fully realize the benefits of novel male contraceptive options, it is incumbent on philanthropic entities, impact investors, venture capital, and/or the pharmaceutical sector to provide significant and timely support for male contraceptive research and development.

Virtual surgical planning and use of a 3D-printed, patient-specific reduction system for minimally invasive plate osteosynthesis of diaphyseal tibial fractures in dogs: A historic case control study.

OBJECTIVE: To compare the efficacy and clinical outcomes of computed tomography (CT)-based virtual surgical planning (VSP) and a three-dimensional (3D)-printed, patient-specific reduction system to conventional indirect reduction techniques for diaphyseal tibial fractures stabilized using minimally invasive plate osteosynthesis (MIPO) in dogs. STUDY DESIGN: A prospective clinical study with a historic control cohort. SAMPLE POPULATION: Dogs undergoing MIPO stabilization of diaphyseal tibial fractures using a custom 3D-printed reduction system (3D-MIPO; n = 15) or conventional indirect reduction techniques (c-MIPO; n = 14). METHODS: Dogs were prospectively enrolled to the 3D-MIPO group and CT scans were used to design and fabricate a custom 3D-printed reduction system to facilitate MIPO. Medical records were searched to identify dogs for the c-MIPO group. Pre-, intra- and postoperative parameters were compared between groups. RESULTS: The duration from presentation until surgery was 23 h longer in the 3D-MIPO group (p = .002). Fewer intraoperative fluoroscopic images were acquired (p < .001) and mean surgical duration was 34 min shorter in the 3D-MIPO group (p = .014). Median postoperative tibial length, frontal alignment, and sagittal alignment were within 4 mm, 3° and 3°, respectively, of the contralateral tibia in both groups and did not differ between reduction groups (p > .1). Postoperative complications occurred in 27% and 14% of fractures in the 3D-MIPO and c-MIPO groups, respectively. CONCLUSION: Both reduction methods yielded comparable results. Although the preoperative planning and guide preparation was time consuming, surgery times were shorter and fluoroscopy use was less in the 3D-MIPO group. CLINICAL SIGNIFICANCE: VSP and the custom 3D-printed reduction system facilitated efficient MIPO.

Rapidly Self-Healable and Melt-Extrudable Polyethylene Reprocessable Network Enabled with Dialkylamino Disulfide Dynamic Chemistry.

Catalyst-free, radical-based reactive processing is used to transform low-density polyethylene (LDPE) into polyethylene covalent adaptable networks (PE CANs) using a dialkylamino disulfide crosslinker, BiTEMPS methacrylate (BTMA). Two versions of BTMA are used, BTMA-S2, with nearly exclusively disulfide bridges, and BTMA-Sn, with a mixture of oligosulfide bridges, to produce S2 PE CAN and Sn PE CAN, respectively. The two PE CANs exhibit identical crosslink densities, but the S2 PE CAN manifests faster stress relaxation, with average relaxation times ∼4.5 times shorter than those of Sn PE CAN over a 130 to 160 °C temperature range. The more rapid dynamics of the S2 PE CAN translate into a shorter compression-molding reprocessing time at 160 °C of only 5 min (vs 30 min for the Sn PE CAN) to achieve full recovery of crosslink density. Both PE CANs are melt-extrudable and exhibit full recovery within experimental uncertainty of crosslink density after extrusion. Both PE CANs are self-healable, with a crack fully repaired and the original tensile properties restored after 30 min for the S2 PE CAN or 60 min for the Sn PE CAN at a temperature slightly above the LDPE melting point and without the assistance of external forces.

Cutibacterium acnes Infection as a Cause of Nonunion After Ulnar-Shortening Osteotomy.

Ulnar-shortening osteotomy is a reliable solution to treat ulnar impaction syndrome, but it has a significant rate of nonunion as a known complication. Generally nonunion after the procedure is attributed to noninfectious causes. When infections happen, they follow the microbiological trends of nonunions elsewhere in the body. We present a case of ulnar-shortening osteotomy using an oblique-cut osteotomy system that resulted in septic nonunion. At the time of revision surgery, Cutibacterium acnes and Staphylococcus hominis were isolated from the osteotomy site. The patient was successfully treated using intravenous antibiotics and the two-stage Masquelet technique and eventually went on to bony union. As C acnes is rarely encountered in this context, this report highlights the need to consider all possible pathogens in the workup of a potentially septic nonunion. Surgeons should consider bacteria such as C acnes that require prolonged incubation for isolation from cultures, which may not be part of many institutions' usual protocol. [Orthopedics. 2024;47(4):e211-e213.].

"Modeling Diffusive Search by Non-Adaptive Sperm: Empirical and Computational Insights".

During fertilization, mammalian sperm undergo a winnowing selection process that reduces the candidate pool of potential fertilizers from ~106-1011 cells to 101-102 cells (depending on the species). Classical sperm competition theory addresses the positive or 'stabilizing' selection that acts on sperm phenotypes within populations of organisms but does not strictly address the developmental consequences of sperm traits among individual organisms that are under purifying selection during fertilization. It is the latter that is of utmost concern for improving assisted reproductive technologies (ART) because 'low fitness' sperm may be inadvertently used for fertilization during interventions that rely heavily on artificial sperm selection, such as intracytoplasmic sperm injection (ICSI). Importantly, some form of sperm selection is used in nearly all forms of ART (e.g., differential centrifugation, swim-up, or hyaluronan binding assays, etc.). To date, there is no unifying quantitative framework (i.e., theory of sperm selection) that synthesizes causal mechanisms of selection with observed natural variation in individual sperm traits. In this report, we reframe the physiological function of sperm as a collective diffusive search process and develop multi-scale computational models to explore the causal dynamics that constrain sperm 'fitness' during fertilization. Several experimentally useful concepts are developed, including a probabilistic measure of sperm 'fitness' as well as an information theoretic measure of the magnitude of sperm selection, each of which are assessed under systematic increases in microenvironmental selective pressure acting on sperm motility patterns.

Physiology, fast and slow: bacterial response to variable resource stoichiometry and dilution rate.

Microorganisms grow despite imbalances in the availability of nutrients and energy. The biochemical and elemental adjustments that bacteria employ to sustain growth when these resources are suboptimal are not well understood. We assessed how Pseudomonas putida KT2440 adjusts its physiology at differing dilution rates (to approximate growth rates) in response to carbon (C), nitrogen (N), and phosphorus (P) stress using chemostats. Cellular elemental and biomolecular pools were variable in response to different limiting resources at a slow dilution rate of 0.12 h-1, but these pools were more similar across treatments at a faster rate of 0.48 h-1. At slow dilution rates, limitation by P and C appeared to alter cell growth efficiencies as reflected by changes in cellular C quotas and rates of oxygen consumption, both of which were highest under P- and lowest under C- stress. Underlying these phenotypic changes was differential gene expression of terminal oxidases used for ATP generation that allows for increased energy generation efficiency. In all treatments under fast dilution rates, KT2440 formed aggregates and biofilms, a physiological response that hindered an accurate assessment of growth rate, but which could serve as a mechanism that allows cells to remain in conditions where growth is favorable. Our findings highlight the ways that microorganisms dynamically adjust their physiology under different resource supply conditions, with distinct mechanisms depending on the limiting resource at slow growth and convergence toward an aggregative phenotype with similar compositions under conditions that attempt to force fast growth. IMPORTANCE: All organisms experience suboptimal growth conditions due to low nutrient and energy availability. Their ability to survive and reproduce under such conditions determines their evolutionary fitness. By imposing suboptimal resource ratios under different dilution rates on the model organism Pseudomonas putida KT2440, we show that this bacterium dynamically adjusts its elemental composition, morphology, pools of biomolecules, and levels of gene expression. By examining the ability of bacteria to respond to C:N:P imbalance, we can begin to understand how stoichiometric flexibility manifests at the cellular level and impacts the flow of energy and elements through ecosystems.

Reprocessable Polymer Networks Containing Sulfur-Based, Percolated Dynamic Covalent Cross-Links and Percolated or Non-Percolated, Static Cross-Links.

One method to improve the properties of covalent adaptable networks (CANs) is to reinforce them with a fraction of permanent cross-links without sacrificing their (re)processability. Here, a simple method to synthesize poly(n-hexyl methacrylate) (PHMA) and poly(n-lauryl methacrylate) (PLMA) networks containing static dialkyl disulfide cross-links (utilizing bis(2-methacryloyl)oxyethyl disulfide, or DSDMA, as a permanent cross-linker) and dynamic dialkylamino sulfur-sulfur cross-links (utilizing BiTEMPS methacrylate as a dissociative dynamic covalent cross-linker) is presented. The robustness and (re)processability of the CANs are demonstrated, including the full recovery of cross-link density after recycling. The authors also investigate the effect of static cross-link content on the stress relaxation responses of the CANs with and without percolated, static cross-links. As PHMA and PLMA have very different activation energies of their respective cooperative segmental mobilities, it is shown that the dissociative CANs without percolated, static cross-links have activation energies of stress relaxation that are dominated by the dissociation of BiTEMPS methacrylate cross-links rather than by the cooperative relaxations of backbone segments, i.e., the alpha relaxation. In CANs with percolated, static cross-links, the segmental relaxation of side chains, i.e., the beta relaxation, is critical in allowing for large-scale stress relaxation and governs their activation energies of stress relaxation.

Risk Factors Associated with Suicidal Ideation and Suicidal Behavior in Wildland Firefighters.

INTRODUCTION: Wildland firefighters are posited to have a higher suicide rate than general firefighters and the general population. However, the rates and risk factors of suicide among wildland firefighters is not fully understood. METHODS: For this preregistered study, 564 participants were recruited from Facebook to obtain a final sample of 246 participants with valid data who were current or former wildland firefighters in the United States. Participants completed online measures of suicidal ideation, history of suicidal behaviors, posttraumatic stress disorder (PTSD) symptoms, problematic alcohol use, and occupational exposure to suicide. RESULTS: In this sample, 22% of wildland firefighters reported a history of at least one suicide attempt, and 36.7% reported current suicidal ideation. PTSD symptoms, but not problematic alcohol use or exposure to suicide, were positively associated with suicidal ideation and a history of suicide attempts. Additionally, PTSD symptoms explained significantly more variance in suicidal ideation than depression symptoms alone. CONCLUSIONS: Wildland firefighters demonstrate rates of suicide attempts that exceed those of non-wildland firefighters and of the general population at large. In addition, PTSD symptoms may contribute to suicidal thoughts and behaviors (STB) in this population. This is the largest study of STB in wildland firefighters to date.

Obesity-driven mitochondrial dysfunction in human adipose tissue-derived mesenchymal stem/stromal cells involves epigenetic changes.

Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms have not been sufficiently elucidated. Mitochondria modulate the viability, plasticity, proliferative capacity, and differentiation potential of MSCs. We hypothesized that alterations in the 5-hydroxymethylcytosine (5hmC) profile of mitochondria-related genes may mediate obesity-driven dysfunction of human adipose-derived MSCs. MSCs were harvested from abdominal subcutaneous fat of obese and age/sex-matched non-obese subjects (n = 5 each). The 5hmC profile and expression of nuclear-encoded mitochondrial genes were examined by hydroxymethylated DNA immunoprecipitation sequencing (h MeDIP-seq) and mRNA-seq, respectively. MSC mitochondrial structure (electron microscopy) and function, metabolomics, proliferation, and neurogenic differentiation were evaluated in vitro, before and after epigenetic modulation. hMeDIP-seq identified 99 peaks of hyper-hydroxymethylation and 150 peaks of hypo-hydroxymethylation in nuclear-encoded mitochondrial genes from Obese- versus Non-obese-MSCs. Integrated hMeDIP-seq/mRNA-seq analysis identified a select group of overlapping (altered levels of both 5hmC and mRNA) nuclear-encoded mitochondrial genes involved in ATP production, redox activity, cell proliferation, migration, fatty acid metabolism, and neuronal development. Furthermore, Obese-MSCs exhibited decreased mitochondrial matrix density, membrane potential, and levels of fatty acid metabolites, increased superoxide production, and impaired neuronal differentiation, which improved with epigenetic modulation. Obesity elicits epigenetic changes in mitochondria-related genes in human adipose-derived MSCs, accompanied by structural and functional changes in their mitochondria and impaired fatty acid metabolism and neurogenic differentiation capacity. These observations may assist in developing novel therapies to preserve the potential of MSCs for tissue repair and regeneration in obese individuals.

HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α-targeted therapies and deplete pathogenically stabilized HIF-2α.

Efficacy of virtual surgical planning and a three-dimensional-printed, patient-specific reduction system to facilitate alignment of diaphyseal tibial fractures stabilized by minimally invasive plate osteosynthesis in dogs: A prospective clinical study.

OBJECTIVE: To evaluate the efficacy of a three-dimensional (3D)-printed, patient-specific reduction system for aligning diaphyseal tibial fractures stabilized using minimally invasive plate osteosynthesis (MIPO). STUDY DESIGN: Prospective clinical trial. SAMPLE POPULATION: Fifteen client owned dogs. METHODS: Virtual 3D models of both pelvic limbs were created. Pin guides were designed to conform to the proximal and distal tibia. A reduction bridge was designed to align the pin guides based on the guides' spatial location. Guides were 3D printed, sterilized, and applied, in conjunction with transient application of a circular fixator, to facilitate indirect fracture realignment before plate application. Alignment of the stabilized tibiae was assessed using postoperative computed tomography scans. RESULTS: Mean duration required for virtual planning was 2.5 h and a mean of 50.7 h elapsed between presentation and surgery. Guide placement was accurate with minor median discrepancies in translation and frontal, sagittal, and axial plane positioning of 2.9 mm, 3.6°, 2.7°, and 6.8°, respectively. Application of the reduction system restored mean tibial length and frontal, sagittal, and axial alignment within 1.7 mm, 1.9°, 1.7°, and 4.5°, respectively, of the contralateral tibia. CONCLUSION: Design and fabrication of a 3D-printed, patient-specific fracture reduction system is feasible in a relevant clinical timeline. Intraoperative pin-guide placement was reasonably accurate with minor discrepancies compared to the virtual plan. Custom 3D-printed reduction system application facilitated near-anatomic or acceptable fracture reduction in all dogs. CLINICAL SIGNIFICANCE: Virtual planning and fabrication of a 3D-printing patient-specific fracture reduction system is practical and facilitated acceptable, if not near-anatomic, fracture alignment during MIPO.

The Open DAC 2023 Dataset and Challenges for Sorbent Discovery in Direct Air Capture.

Direct air capture (DAC) of CO2 with porous adsorbents such as metal-organic frameworks (MOFs) has the potential to aid large-scale decarbonization. Previous screening of MOFs for DAC relied on empirical force fields and ignored adsorbed H2O and MOF deformation. We performed quantum chemistry calculations overcoming these restrictions for thousands of MOFs. The resulting data enable efficient descriptions using machine learning.

Hepatocyte-Specific PEX16 Abrogation in Mice Leads to Hepatocyte Proliferation, Alteration of Hepatic Lipid Metabolism, and Resistance to High-Fat Diet (HFD)-Induced Hepatic Steatosis and Obesity.

Obesity results in hepatic fat accumulation, i.e., steatosis. In addition to fat overload, impaired fatty acid ß-oxidation also promotes steatosis. Fatty acid ß-oxidation takes place in the mitochondria and peroxisomes. Usually, very long-chain and branched-chain fatty acids are the first to be oxidized in peroxisomes, and the resultant short chain fatty acids are further oxidized in the mitochondria. Peroxisome biogenesis is regulated by peroxin 16 (PEX16). In liver-specific PEX16 knockout (Pex16Alb-Cre) mice, hepatocyte peroxisomes were absent, but hepatocytes proliferated, and liver mass was enlarged. These results suggest that normal liver peroxisomes restrain hepatocyte proliferation and liver sizes. After high-fat diet (HFD) feeding, body weights were increased in PEX16 floxed (Pex16fl/fl) mice and adipose-specific PEX16 knockout (Pex16AdipoQ-Cre) mice, but not in the Pex16Alb-Cre mice, suggesting that the development of obesity is regulated by liver PEX16 but not by adipose PEX16. HFD increased liver mass in the Pex16fl/fl mice but somehow reduced the already enlarged liver mass in the Pex16Alb-Cre mice. The basal levels of serum triglyceride, free fatty acids, and cholesterol were decreased, whereas serum bile acids were increased in the Pex16Alb-Cre mice, and HFD-induced steatosis was not observed in the Pex16Alb-Cre mice. These results suggest that normal liver peroxisomes contribute to the development of liver steatosis and obesity.

Mechanistic study of the stabilization of dentin-bonded restorative interfaces via collagen reinforcement by multi-acrylamides.

Hydrolytically and enzymatically-stable multi-acrylamides have been proposed to increase the long-term durability of dental adhesive interfaces as alternatives to methacrylates. The aim of this study was to investigate the mechanical and biochemical properties of experimental adhesives containing multi-functional acrylamides concerning collagen reinforcement and metalloproteinases (MMP) activity. Multi-functional acrylamides, TMAAEA (Tris[(2-methylaminoacryl) ethylamine) and DEBAAP (N,N-Diethyl-1,3-bis(acrylamido) propane), along with the commercially available DMAM (N,N-dimethylacrylamide) (monofunctional acrylamide) and HEMA (2-Hydroxyethyl methacrylate) (monofunctional methacrylate - control) were tested for stability against enzymatic hydrolysis by cholesterol esterase/pseudocholinesterase (PC/PCE) solutions for up to 30 days. Collagen-derived substrate and gelatin zymography were performed to examine the effect of the compounds on the biological activity of human recombinant and dentin-extracted gelatinases MMP-2 and MMP-9. In situ zymography was carried out by fluorescent collagen degradation combined with confocal microscopy analysis. Hydroxyproline content was measured in collagen derived from dentin extracts though reaction with Ehrlich's reagent p-dimethylaminobenzaldehyde (DMAB), generating a stable chromophore measured at 550 nm. Storage shear modulus of demineralized dentin discs treated with the tested compounds was measured by oscillatory rheometry, in order to investigate potential collagen reinforcement. FT-IR was performed to determine qualitative differences in collagen based on observed changes in amide bands. The results were analyzed by ANOVA/Tukey's test (α = 0.05). Multi-acrylamides survived 30 days of incubation in cholinesterase/pseudo-cholinesterase (PC/PCE) solutions, while HEMA showed approximately 70 % overall degradation. Incubation with multi-acrylamides reduced collagen degradation as evidenced by the reduced hydroxyproline levels and by the 30 % increase inshear storage modulus. Biochemical and zymography assays showed no noticeable inhibition of recombinant and extracted MMPs enzymatic activity. The infra-red spectroscopy results for multi-functional acrylamides treated samples demonstrated shifts of the amide II bonds and marked increase in intensity of the bands 1200 cm-1, which may indicate partial collagen denaturation and some degree of cross-linking of the compounds with collagen, respectively. The multi-acrylamides exhibited not only comparable mechanical properties but also demonstrated significantly enhanced biochemical stability when compared to the widely used methacrylate control. Clinical relevance: These findings highlight the potential of multi-acrylamides to increase the bonding stability to tissues and, ultimately, contribute to the longevity of dental restorations.

Conjoint tendon lengthening improves internal rotation following reverse total shoulder arthroplasty: a cadaveric study.

BACKGROUND: Reverse shoulder arthroplasty (RSA) is a common procedure for treating a variety of shoulder pathologies. However, many patients struggle with postoperative internal rotation (IR) deficits, which often hinder their activities of daily living. The conjoint tendon provides an anatomic barrier that can impede the postoperative IR of the shoulder, and this study aims to evaluate the effect of a conjoint tendon lengthening on the glenohumeral range of motion (ROM) following RSA. METHODS: This study used ten fresh-frozen cadaver specimens of the upper extremity. An RSA was implanted using a standard deltopectoral approach, and the ROM was assessed postimplantation. Following this, the conjoint tendon was identified and lengthened using a tendon sheath z-plasty, and the ROM was rerecorded. Statistical significance for the ROM gains after conjoint tendon lengthening was determined with a significance level of P < .05. RESULTS: Following the lengthening of the conjoint tendon, there were statistically significant improvements in all ROMs (P < .05). Subjects demonstrated a notable gain in IR to the back by 10.3 cm (P < .01), and all ROMs increased by at least 10°, except for forward flexion, which increased by 6° (P < .001). CONCLUSIONS: This study suggests that lengthening the conjoint tendon improves postoperative ROM of the glenohumeral joint after RSA, offering a potential solution to considerable IR deficits that are commonly encountered post-RSA. Subsequent clinical and biomechanical studies should assess the stability of the shoulder joint following conjoint tendon lengthening.

Real-time identification of life-threatening necrotizing soft-tissue infections using indocyanine green fluorescence imaging.

Significance: Necrotizing soft-tissue infections (NSTIs) are life-threatening infections with a cumulative case fatality rate of 21%. The initial presentation of an NSTI is non-specific, frequently leading to misdiagnosis and delays in care. No current strategies yield an accurate, real-time diagnosis of an NSTI. Aim: A first-in-kind, observational, clinical pilot study tested the hypothesis that measurable fluorescence signal voids occur in NSTI-affected tissues following intravenous administration and imaging of perfusion-based indocyanine green (ICG) fluorescence. This hypothesis is based on the established knowledge that NSTI is associated with local microvascular thrombosis. Approach: Adult patients presenting to the Emergency Department of a tertiary care medical center at high risk for NSTI were prospectively enrolled and imaged with a commercial fluorescence imager. Single-frame fluorescence snapshot and first-pass perfusion kinetic parameters-ingress slope (IS), time-to-peak (TTP) intensity, and maximum fluorescence intensity (IMAX)-were quantified using a dynamic contrast-enhanced fluorescence imaging technique. Clinical variables (comorbidities, blood laboratory values), fluorescence parameters, and fluorescence signal-to-background ratios (SBRs) were compared to final infection diagnosis. Results: Fourteen patients were enrolled and imaged (six NSTI, six cellulitis, one diabetes mellitus-associated gangrene, and one osteomyelitis). Clinical variables demonstrated no statistically significant differences between NSTI and non-NSTI patient groups (p-value≥0.22). All NSTI cases exhibited prominent fluorescence signal voids in affected tissues, including tissue features not visible to the naked eye. All cellulitis cases exhibited a hyperemic response with increased fluorescence and no distinct signal voids. Median lesion-to-background tissue SBRs based on snapshot, IS, TTP, and IMAX parameter maps ranged from 3.2 to 9.1, 2.2 to 33.8, 1.0 to 7.5, and 1.5 to 12.7, respectively, for the NSTI patient group. All fluorescence parameters except TTP demonstrated statistically significant differences between NSTI and cellulitis patient groups (p-value<0.05). Conclusions: Real-time, accurate discrimination of NSTIs compared with non-necrotizing infections may be possible with perfusion-based ICG fluorescence imaging.

"A Low-Cost Microfluidics System for Light Microscopy Experiments".

Microfluidics devices are powerful tools for studying dynamic processes in live cells, especially when used in conjunction with light microscopy. There are many applications of microfluidics devices including recording dynamic cellular responses to small molecules or other chemical conditions in perfused media, monitoring cell migration in constrained spaces, or collecting media perfusate for the study of secreted compounds in response to experimental inputs/manipulations. Here we describe a configurable low-cost (channel-based) microfluidics platform for live-cell microscopy, intended to be useful for experiments that require more precision/flexibility than simple rubber spacers, but less precision than molded elastomer-based platforms. The materials are widely commercially available, low-cost, and device assembly takes only minutes.

Three-dimensionally printed osteotomy and reaming guides for correction of a multiplanar femoral deformity stabilized with an interlocking nail in a dog.

OBJECTIVE: To describe the use of virtual surgical planning (VSP) and three-dimensionally (3D) printed surgical guides for corrective osteotomies stabilized with an interlocking nail in a dog with a multiplanar femoral deformity. STUDY DESIGN: Case report. ANIMALS: An 8-year-old male neutered mixed breed dog weighing 44 kg. METHODS: A dog was presented for a right grade 3 lateral patellar luxation secondary to a multiplanar femoral deformity due to a suspected femoral malunion. A computed tomography (CT) scan was obtained to create virtual femoral models. Corrective osteotomies were simulated with VSP. Custom osteotomy guides and reaming guides were designed to facilitate the correction and the placement of an interlocking nail. The preoperative femoral model, virtually aligned femoral model, custom osteotomy guides, and reaming guides were 3D printed, sterilized, and utilized intraoperatively. A CT scan was performed postoperatively to assess femoral length and alignment. RESULTS: Custom osteotomy and reaming guides were used as intended by the VSP. Postoperative femoral length as well as frontal, sagittal, and axial plane alignment were within 0.7 mm, 2.2°, 0.5°, and 1.6°, respectively, of the virtually planned femoral model. Two months postoperatively, the dog was sound on visual gait examination, and the patella tracked in the trochlear groove throughout stifle range of motion and was unable to be manually luxated. Radiographs obtained 2 months postoperatively revealed static femoral alignment and implants. Both osteotomies were discernable with callus bridging. CONCLUSION: Virtual surgical planning and custom osteotomy and reaming guides facilitated complex femoral corrective osteotomies and interlocking nail placement.

Development of quantitative high-throughput screening assays to identify, validate, and optimize small-molecule stabilizers of misfolded ß-glucocerebrosidase with therapeutic potential for Gaucher disease and Parkinson's disease.

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small pro-luminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and non-inhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: the fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 provided direct visualization of GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy, by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically-relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of new chemical matter targeting GCase, ultimately leading to a viable therapeutic for two protein-misfolding diseases.

Ecological characteristics explain neutral genetic variation of three coastal sparrow species.

Eco-phylogeographic approaches to comparative population genetic analyses allow for the inclusion of intrinsic influences as drivers of intraspecific genetic structure. This insight into microevolutionary processes, including changes within a species or lineage, provides better mechanistic understanding of species-specific interactions and enables predictions of evolutionary responses to environmental change. In this study, we used single nucleotide polymorphisms (SNPs) identified from reduced representation sequencing to compare neutral population structure, isolation by distance (IBD), genetic diversity and effective population size (Ne) across three closely related and co-distributed saltmarsh sparrow species differing along a specialization gradient-Nelson's (Ammospiza nelsoni subvirgata), saltmarsh (A. caudacuta) and seaside sparrows (A. maritima maritima). Using an eco-phylogeographic lens within a conservation management context, we tested predictions about species' degree of evolutionary history and ecological specialization to tidal marshes, habitat, current distribution and population status on population genetic metrics. Population structure differed among the species consistent with their current distribution and habitat factors, rather than degree of ecological specialization: seaside sparrows were panmictic, saltmarsh sparrows showed hierarchical structure and Nelson's sparrows were differentiated into multiple, genetically distinct populations. Neutral population genetic theory and demographic/evolutionary history predicted patterns of genetic diversity and Ne rather than degree of ecological specialization. Patterns of population variation and evolutionary distinctiveness (Shapely metric) suggest different conservation measures for long-term persistence and evolutionary potential in each species. Our findings contribute to a broader understanding of the complex factors influencing genetic variation, beyond specialist-generalist status and support the role of an eco-phylogeographic approach in population and conservation genetics.