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Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale. Moreover, as the analysis is based on the accurate measurement of an intact protein, top-down mass spectrometry opens a research paradigm to perform quantitative analysis of \"unknown\" proteins that differ in accurate mass. As a proof of concept, we have applied differential mass spectrometry (dMS) to the top-down analysis of apolipoproteins isolated from human HDL₃. The protein species at 9415.45 Da demonstrates an average fold change of 4.7 (p-value 0.017) and was identified as an O-glycosylated form of apolipoprotein C-III [NANA-(2 [rightward arrow] 3)-Gal-β(1 [rightward arrow] 3)-GalNAc, +656.2037 Da], a protein associated with coronary artery disease. This work demonstrates the utility of top-down dMS for quantitative analysis of intact protein mixtures and holds potential for facilitating a better understanding of HDL biology and complex biological systems at the protein level.

Label-free LC-MS profiling is a powerful quantitative proteomic method to study relative peptide abundances between two or more biological samples. Here we demonstrate the use of a previously described comparative LC-MS method, differential mass spectrometry (dMS), to analyze high-resolution Fourier transform mass spectrometry (FTMS) data for detection and quantification of known peptide differences between two sets of complex mixtures. Six standard peptides were spiked into a processed plasma background at fixed ratios from 1.25:1 to 4:1 to make two sets of samples. The resulting mixtures were analyzed by microcapillary LC-FTMS and dMS. dMS successfully identified five out of the six peptides as statistically significant differences ( p ≤ 0.005). In this experiment, the smallest fold change reliably detected by our method was 1.5:1, and the errors of estimated ratios of concentrations were less than 20% for peptides spiked at 1.5:1 to 4:1. We conclude that LC-FTMS coupled with dMS is a useful label-free quantitative MS method that can be used to detect subtle yet statistically significant peptide differences in complex protein mixtures, including plasma samples.

Disease modifying treatments for Alzheimer's disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle. Cerebrospinal fluid (CSF) biochemical markers such as total tau, p-tau and Ab42 are well established markers of AD; however, global quantitative biochemical changes in CSF in AD disease progression remain largely uncharacterized. Here we applied a high resolution open discovery platform, dMS, to profile a cross-sectional cohort of lumbar CSF from post-mortem diagnosed AD patients versus those from non-AD/non-demented (control) patients. Multiple markers were identified to be statistically significant in the cohort tested. We selected two markers SME-1 (p<0.0001) and SME-2 (p = 0.0004) for evaluation in a second independent longitudinal cohort of human CSF from post-mortem diagnosed AD patients and age-matched and case-matched control patients. In cohort-2, SME-1, identified as neuronal secretory protein VGF, and SME-2, identified as neuronal pentraxin receptor-1 (NPTXR), in AD were 21% (p = 0.039) and 17% (p = 0.026) lower, at baseline, respectively, than in controls. Linear mixed model analysis in the longitudinal cohort estimate a decrease in the levels of VGF and NPTXR at the rate of 10.9% and 6.9% per year in the AD patients, whereas both markers increased in controls. Because these markers are detected by mass spectrometry without the need for antibody reagents, targeted MS based assays provide a clear translation path for evaluating selected AD disease-progression markers with high analytical precision in the clinic.

Recent data suggest that cystic fibrosis transmembrane conductance regulator (CFTR) gene alterations negatively impact male fertility beyond obstruction. We sought to compare gene alterations, sperm retrieval rates, and intracytoplasmic sperm injection (ICSI) outcomes among men with cystic fibrosis (CF) disease and congenital bilateral absence of the vas deferens (CBAVD) only. We retrospectively evaluated all men who underwent surgical sperm retrieval at two academic, high-volume andrology centers from 2010 to 2018. Only men with documented CFTR alterations and obstructive azoospermia from either CBAVD or CF were included. Differences between groups for CFTR abnormality, sperm retrieval, and ICSI outcomes were statistically analyzed. Overall, 39 patients were included with 10 in the CF and 29 in the CBAVD groups. Surgical sperm retrieval rates were significantly lower in the CF group for sperm concentration (14.8 × 10[6] ml-1 vs 61.4 × 10[6] ml−1, P = 0.02) and total motile sperm count (2.9 million vs 11.4 million, P = 0.01). This difference was only predicted by homozygous delta F508 CFTR mutations (P < 0.05). The CF group also demonstrated a significantly higher rate of rescue testicular sperm extraction (70.0% vs 27.6%, P < 0.03) and lower fertilization rate with ICSI (32.5% vs 68.9%, P < 0.01). In conclusion, those with CF demonstrated lower sperm quality, greater difficulty with sperm retrieval, and worse ICSI outcomes compared with CBAVD-only patients. Homozygous delta F508 CFTR mutations appear to significantly impair spermatogenesis and sperm function.

Purpose Additive manufacture (AM) such as selective laser melting (SLM) provides significant geometric design freedom in comparison with traditional manufacturing methods. Such freedom enables the construction of injection moulding tools with conformal cooling channels that optimize heat transfer while incorporating efficient internal lattice structures that can ground loads and provide thermal insulation. Despite the opportunities enabled by AM, there remain a number of design and processing uncertainties associated with the application of SLM to injection mould tool manufacture, in particular from H13/DIN 1.2344 steel as commonly used in injection moulds. This paper aims to address several associated uncertainties. Design/methodology/approach A number of physical and numerical experimental studies are conducted to quantify SLM-manufactured H13 material properties, part manufacturability and part characteristics. Findings Findings are presented which quantify the effect of SLM processing parameters on the density of H13 steel components; the manufacturability of standard and self-supporting conformal cooling channels, as well as structural lattices in H13; the surface roughness of SLM-manufactured cooling channels; the effect of cooling channel layout on the associated stress concentration factor and cooling uniformity; and the structural and thermal insulating properties of a number of structural lattices. Originality/value The contributions of this work with regards to SLM manufacture of H13 of injection mould tooling can be applied in the design of conformal cooling channels and lattice structures for increased thermal performance.

As the prevalence of diabetes is increasing worldwide, research on some of the lesser-known effects, including impaired bone health, are gaining a lot of attention. The two most common forms of diabetes are type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). These two differ in their physiology, with T1DM stemming from an inability to produce insulin, and T2DM involving an insufficient response to the insulin that is produced. This review aims to highlight the most current information regarding diabetes as it relates to bone health. It looks at biochemical changes that characterize diabetic bone; notably increased adiposity, altered bone metabolism, and variations in bone mineral density (BMD). Then several hypotheses are analyzed, concerning how these changes may be detrimental to the highly orchestrated processes that are involved in bone formation and turnover, and ultimately result in the distinguishing features of diabetic bone. The review proceeds by explaining the effects of antidiabetes medications on bone health, then highlighting several ways that diabetes can play a part in other clinical treatment outcomes. With diabetes negatively affecting bone health and creating other clinical problems, and its treatment options potentiating these effects, physicians should consider the use of anti-osteoporotic drugs to supplement standard anti-diabetes medications in patients suffering with diabetic bone loss.

Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.

Each human spermatozoon contains two remodeled centrioles that it contributes to the zygote. There, the centrioles reconstitute a centrosome that assembles the sperm aster and participate in pronuclei migration and cleavage. Thus, centriole abnormalities may be a cause of male factor infertility and failure to carry pregnancy to term. However, the precise mechanisms by which sperm centrioles contribute to embryonic development in humans are still unclear, making the search for a link between centriole abnormalities and impaired male fecundity particularly difficult. Most previous investigations into the role of mammalian centrioles during fertilization have been completed in murine models; however, because mouse sperm and zygotes appear to lack centrioles, these studies provide information that is limited in its applicability to humans. Here, we review studies that examine the role of the sperm centrioles in the early embryo, with particular emphasis on humans. Available literature includes case studies and case-control studies, with a few retrospective studies and no prospective studies reported. This literature has provided some insight into the morphological characteristics of sperm centrioles in the zygote and has allowed identification of some centriole abnormalities in rare cases. Many of these studies suggest centriole involvement in early embryogenesis based on phenotypes of the embryo with only indirect evidence for centriole abnormality. Overall, these studies suggest that centriole abnormalities are present in some cases of sperm with asthenoteratozoospermia and unexplained infertility. Yet, most previously published studies have been restricted by the laborious techniques (like electron microscopy) and the limited availability of centriolar markers, resulting in small-scale studies and the lack of solid causational evidence. With recent progress in sperm centriole biology, such as the identification of the unique composition of sperm centrioles and the discovery of the atypical centriole, it is now possible to begin to fill the gaps in sperm centriole epidemiology and to identify the etiology of sperm centriole dysfunction in humans.

Nipah virus (NiV), a highly pathogenic zoonotic paramyxovirus, causes severe respiratory and neurological disease in humans, with a case-fatality rate around 60%. Descriptions of cases in the clinical setting suggest that the two primary lineages of NiV cause disease with different presentations and outcomes. To define strain-specific differences in disease progression and host responses, African green monkeys were exposed to either the Malaysia (NiV-M) or Bangladesh (NiV-B) strain using a large-particle aerosol exposure. NiV-M infection resulted in a fatality rate of 27%, while NiV-B infection led to a 75% fatality rate characterized by rapid respiratory decline and systemic viral dissemination. Among survivors, NiV-M–infected animals mounted robust immunoglobulin M, immunoglobulin G, and neutralizing antibody responses, whereas NiV-B survivors exhibited weaker and delayed humoral responses. Non-survivors of both strains showed elevated pro-inflammatory cytokines, thrombocytopenia, and multi-organ dysfunction. Imaging showed that NiV-M infection was associated with neuroinflammation and systemic vasculitis, while NiV-B infection caused progressive pulmonary pathology. Histopathological analysis confirmed widespread vasculitis and encephalitis in animals with NiV-M infection and diffuse pulmonary hemorrhage and fibrin thrombi, consistent with vascular injury and coagulopathy, in animals with NiV-B infection. Cytokine profiling and flow cytometry showed a more intense and dysregulated immune response to NiV-B infection. Fatal outcomes in both groups were associated with thrombocytopenia, elevated pro-inflammatory cytokines, and multi-organ dysfunction. This study highlights fundamental differences in virulence, immune evasion, and pathogenesis between NiV strains and underscores the value of the African green monkey aerosol model for evaluating medical countermeasures under conditions that closely mimic natural human exposure.

Background and Objectives: Femoral malunion, defined as healing of a femoral fracture in an anatomically incorrect position, can lead to significant biomechanical and functional impairment despite modern fixation techniques achieving union rates near 99%. The lack of a universal definition and standardized management approach continues to hinder optimal outcomes. This review aims to synthesize the literature on the causes, clinical presentation, radiologic assessment, surgical indications, corrective procedures, and outcomes of femoral malunion to guide clinical decision-making and future research. Materials and Methods: A narrative review of peer-reviewed orthopedic literature was conducted, focusing on adult femoral malunions across anatomical regions. Articles detailing deformity thresholds, imaging modalities, corrective osteotomies, and fixation strategies were included. Particular emphasis was placed on region-specific deformities—femoral head, neck, intertrochanteric, diaphyseal, and distal femur—and their corresponding surgical correction methods, including valgus intertrochanteric osteotomy, clamshell osteotomy, and lengthening with external or magnetic intramedullary devices. Results: Malunion most commonly presents as angular, rotational, or length deformity, with thresholds of >5–10° angulation, >10° rotation, or >1–2 cm shortening being clinically significant. Patients may experience pain, limp, gait asymmetry, and early-onset arthritis. Corrective techniques tailored to the anatomical site yield favorable results: valgus intertrochanteric osteotomy restores leg length and alignment; diaphyseal malunions respond well to single- or multi-plane osteotomies with internal fixation or gradual correction; distal femoral malunions often require multiplanar osteotomy to reestablish the joint line. Most series report high union rates and functional improvement, though complications such as infection and hardware failure may occur. Conclusions: Femoral malunion remains a complex but treatable condition. Successful outcomes rely on accurate deformity characterization, patient-specific surgical planning, and restoration of mechanical alignment. Standardized deformity criteria and long-term functional outcome studies are needed to refine management algorithms and improve patient care.