Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity.

Our data suggest that, after a myocardial infarction, integrin-associated protein CD47 on cardiac myocytes is elevated. In culture, increased CD47 on the surface of dying cardiomyocytes impairs phagocytic removal by immune cell macrophages. After myocardial ischemia and reperfusion, acute CD47 inhibition with blocking antibodies enhanced dead myocyte clearance by cardiac phagocytes and also improved the resolution of cardiac inflammation, reduced infarct size, and preserved cardiac contractile function. Early targeting of CD47 in the myocardium after reperfusion may be a new strategy to enhance wound repair in the ischemic heart.

Treatment of articular cartilage injuries remains a clinical challenge, and the optimal tools to monitor and predict clinical outcomes are unclear. Quantitative magnetic resonance imaging (qMRI) allows for a noninvasive biochemical evaluation of cartilage and may offer advantages in monitoring outcomes after cartilage repair surgery.

Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors. EC2629 treatment of nude mice bearing FR-positive KB human xenografts led to cures in 100% of the test animals with very low dose levels (300 nmol/kg) following a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing). Complete responses were also observed against FR-positive paclitaxel (KB-PR) and cisplatin (KB-CR) resistant models. When evaluated against FR-positive patient derived xenograft (PDX) models of ovarian (ST070), endometrial (ST040) and triple negative breast cancers (ST502, ST738), EC2629 showed significantly greater anti-tumor activity compared to their corresponding standard of care treatments. Taken together, these studies thus demonstrated that EC2629, with its distinct DNA reacting mechanism, may be useful in treating FR-positive tumors, including those that are classified as drug resistant.

Human Papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) is one of the fastest growing cancers in the Western world. When compared to OPSCCs induced by smoking or alcohol, patients with HPV+ OPSCC, have better survival and the mechanisms remain unclear.

Chlamydia is an obligate intracellular bacterium and the most common reportable cause of human infection in the United States. This pathogen proliferates inside a eukaryotic host cell, where it resides within a membrane-bound compartment called the chlamydial inclusion. It has an unusual developmental cycle, marked by conversion between a replicating form, the reticulate body (RB), and an infectious form, the elementary body (EB). We found that the small molecule H89 slowed inclusion growth and decreased overall RB replication by 2-fold but caused a 25-fold reduction in infectious EBs. This disproportionate effect on EB production was mainly due to a defect in RB-to-EB conversion and not to the induction of chlamydial persistence, which is an altered growth state. Although H89 is a known inhibitor of specific protein kinases and vesicular transport to and from the Golgi apparatus, it did not cause these anti-chlamydial effects by blocking protein kinase A or C or by inhibiting protein or lipid transport. Thus, H89 is a novel anti-chlamydial compound that has a unique combination of effects on an intracellular Chlamydia infection.

Traumatic brain injury (TBI) is a heterogeneous disease in its origin, neuropathology, and prognosis, with no FDA-approved treatments. The pathology of TBI is complicated and not sufficiently understood, which is the reason why more than 30 clinical trials in the past three decades turned out unsuccessful in phase III. The multifaceted pathophysiology of TBI involves a cascade of metabolic and molecular events including inflammation, oxidative stress, excitotoxicity, and mitochondrial dysfunction. In this study, an open head TBI mouse model, induced by controlled cortical impact (CCI), was used to investigate the chronic protective effects of mitoquinone (MitoQ) administration 30 days post-injury. Neurological functions were assessed with the Garcia neuroscore, pole climbing, grip strength, and adhesive removal tests, whereas cognitive and behavioral functions were assessed using the object recognition, Morris water maze, and forced swim tests. As for molecular effects, immunofluorescence staining was conducted to investigate microgliosis, astrocytosis, neuronal cell count, and axonal integrity. The results show that MitoQ enhanced neurological and cognitive functions 30 days post-injury. MitoQ also decreased the activation of astrocytes and microglia, which was accompanied by improved axonal integrity and neuronal cell count in the cortex. Therefore, we conclude that MitoQ has neuroprotective effects in a moderate open head CCI mouse model by decreasing oxidative stress, neuroinflammation, and axonal injury.

Mitochondrial complex I can produce large quantities of reactive oxygen species (ROS) by reverse electron transfer (RET) from the ubiquinone (UQ) pool. Glutathionylation of complex I does induce increased mitochondrial superoxide/hydrogen peroxide (O2●-/H2O2) production, but the source of this ROS has not been identified. Here, we interrogated the glutathionylation of complex I subunit NDUFS1 and examined if its modification can result in increased ROS production during RET from the UQ pool. We also assessed glycerol-3-phosphate dehydrogenase (GPD) and proline dehydrogenase (PRODH) glutathionylation since both flavoproteins have measurable rates for ROS production as well. Induction of glutathionylation with disulfiram induced a significant increase in O2●-/H2O2 production during glycerol-3-phosphate (G3P) and proline (Pro) oxidation. Treatment of mitochondria with inhibitors for complex I (rotenone and S1QEL), complex III (myxothiazol and S3QEL), glycerol-3-phosphate dehydrogenase (iGP), and proline dehydrogenase (TFA) confirmed that the sites for this increase were complexes I and III, respectively. Treatment of liver mitochondria with disulfiram (50-1000 nM) did not induce GPD or PRODH glutathionylation, nor did it affect their activities, even though disulfiram dose-dependently increased the total number of protein glutathione mixed disulfides (PSSG). Immunocapture of complex I showed disulfiram incubations resulted in the modification of NDUFS1 subunit in complex I. Glutathionylation could be reversed by reducing agents, restoring the deglutathionylated state of NDUFS1 and the activity of the complex. Reduction of glutathionyl moieties in complex I also significantly decreased ROS production by RET from GPD and PRODH. Overall, these findings demonstrate that the modification of NDUFS1 can result in increased ROS production during RET from the UQ pool, which has implications for understanding the relationship between mitochondrial glutathionylation reactions and induction of oxidative distress in several pathologies.

This study addresses the challenge of accurately identifying filamentous fungi in medical laboratories using transfer learning with convolutional neural networks (CNNs). The study uses microscopic images from touch-tape slides with lactophenol cotton blue staining, the most common method in clinical settings, to classify fungal genera and identify Aspergillus species. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently. IMPORTANCE This study utilizes transfer learning with CNNs to classify fungal genera and identify Aspergillus species using microscopic images from touch-tape preparation and lactophenol cotton blue staining. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently.

Pancreatic cancer is a highly lethal disease with limited treatment options. Hence, there is a considerable medical need for novel treatment strategies. Monoclonal antibodies (mAbs) have significantly improved cancer therapy, primarily due to their ability to stimulate antibody-dependent cellular cytotoxicity (ADCC), which plays a crucial role in their therapeutic efficacy. As a result, significant effort has been focused on improving this critical function by engineering mAbs with Fc regions that have increased affinity for the Fc receptor CD16 expressed on natural killer (NK) cells, the major cell population that mediates ADCC in humans. Here we report on the preclinical characterization of a mAb directed to the target antigen B7-H3 (CD276) containing an Fc part with the amino acid substitutions S239D/I332E to increase affinity for CD16 (B7-H3-SDIE) for the treatment of pancreatic cancer. B7-H3 (CD276) is highly expressed in many tumor entities, whereas expression on healthy tissues is more limited. Our findings confirm high expression of B7-H3 on pancreatic cancer cells. Furthermore, our study shows that B7-H3-SDIE effectively activates NK cells against pancreatic cancer cells in an antigen-dependent manner, as demonstrated by the analysis of NK cell activation, degranulation and cytokine release. The activation of NK cells resulted in significant tumor cell lysis in both short-term and long-term cytotoxicity assays. In conclusion, B7-H3-SDIE constitutes a promising agent for the treatment of pancreatic cancer.

A common haplotype on 10q26 influences the risk of age-related macular degeneration (AMD) and encompasses two genes, LOC387715 and HTRA1. Recent data have suggested that loss of LOC387715, mediated by an insertion/deletion (in/del) that destabilizes its message, is causally related with the disorder. Here we show that loss of LOC387715 is insufficient to explain AMD susceptibility, since a nonsense mutation (R38X) in this gene that leads to loss of its message resides in a protective haplotype. At the same time, the common disease haplotype tagged by the in/del and rs11200638 has an effect on the transcriptional upregulation of the adjacent gene, HTRA1. These data implicate increased HTRA1 expression in the pathogenesis of AMD and highlight the importance of exploring multiple functional consequences of alleles in haplotypes that confer susceptibility to complex traits.

Age-related macular degeneration (AMD) is the most common cause of irreversible central vision loss worldwide. Research has linked AMD susceptibility with dysregulation of the complement cascade. Typically, complement factor H (CFH), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3) are associated with AMD. In this paper, we investigated the association between complement factor D (CFD), another factor of the complement system, and advanced AMD in a Caucasian population.

The close physical proximity between the Golgi and the centrosome is a unique feature of mammalian cells that has baffled scientists for years. Several knockdown and overexpression studies have linked the spatial relationship between these two organelles to the control of directional protein transport, directional migration, ciliogenesis and mitotic entry. However, most of these conditions have not only separated these two organelles, but also caused extensive fragmentation of the Golgi, making it difficult to dissect the specific contribution of Golgi-centrosome proximity. In this study, we present our results with stable retinal pigment epithelial (RPE-1) cell lines in which GM130 was knocked out using a CRISPR/Cas9 approach. While Golgi and centrosome organization appeared mostly intact in cells lacking GM130, there was a clear separation of these organelles from each other. We show that GM130 may control Golgi-centrosome proximity by anchoring AKAP450 to the Golgi. We also provide evidence that the physical proximity between these two organelles is dispensable for protein transport, cell migration, and ciliogenesis. These results suggest that Golgi-centrosome proximity per se is not necessary for the normal function of RPE-1 cells.

Magnetic resonance imaging (MRI) carries prognostic importance after traumatic brain injury (TBI), especially when computed tomography (CT) fails to fully explain the level of unconsciousness. However, in critically ill patients, the risk of deterioration during transfer needs to be balanced against the benefit of detecting prognostically relevant information on MRI. We therefore aimed to assess if day of injury serum protein biomarkers could identify critically ill TBI patients in whom the risks of transfer are compensated by the likelihood of detecting management-altering neuroimaging findings.

Given its role as the source of definitive hematopoietic cells, we sought to determine whether mutations initiated in the hemogenic endothelium would yield hematopoietic abnormalities or malignancies. Here, we find that endothelium-specific transposon mutagenesis in mice promotes hematopoietic pathologies that are both myeloid and lymphoid in nature. Frequently mutated genes included previously recognized cancer drivers and additional candidates, such as Pi4ka, a lipid kinase whose mutation was found to promote myeloid and erythroid dysfunction. Subsequent validation experiments showed that targeted inactivation of the Pi4ka catalytic domain or reduction in mRNA expression inhibited myeloid and erythroid cell differentiation in vitro and promoted anemia in vivo through a mechanism involving deregulation of AKT, MAPK, SRC, and JAK-STAT signaling. Finally, we provide evidence linking PI4KAP2, previously considered a pseudogene, to human myeloid and erythroid leukemia.

sRNAs are noncoding transcripts that play critical roles in posttranscriptional regulation in prokaryotes. In the intracellular bacterium Chlamydia, sRNAs have been identified, but functional studies have been limited to an E. coli heterologous system. We have developed an inducible sRNA overexpression system in Chlamydia trachomatis and used it to screen putative sRNAs for effects on the Chlamydia developmental cycle, which involves conversion between replicating (RB) and infectious (EB) chlamydial forms. Overexpression of 4 of 13 C. trachomatis sRNAs decreased production of infectious EBs. We performed detailed characterization of CtrR3 and CtrR7, the two sRNAs that caused the largest progeny defects in our screen. By quantifying chlamydial number and infectious progeny, and by visualizing chlamydial forms using electron microscopy, we showed that overexpression of CtrR3 prevented RB-to-EB conversion, whereas CtrR7 overexpression blocked bacterial replication. We also describe a workflow that allowed us to identify the mRNA targets of CtrR3 in Chlamydia. We first used MS2 aptamer affinity purification coupled with RNA sequencing as an unbiased approach to isolate interacting mRNAs. We then prioritized candidates based on sequence complementarity to the CtrR3 target recognition sequence, which we had identified with bioinformatic and mutational analyses. Finally, we tested putative targets with translational fusion assays in E. coli and C. trachomatis. Using this integrated approach, we provide experimental evidence that YtgB and CTL0389 are mRNA targets of CtrR3 in Chlamydia. These findings demonstrate how our C. trachomatis sRNA overexpression system can be used to investigate the functions and mRNA targets of chlamydial sRNAs. IMPORTANCE Small RNAs (sRNAs) are a class of regulatory RNAs that play important roles in bacterial physiology and pathogenesis. In the intracellular bacterium Chlamydia, however, sRNAs are poorly understood, and functional studies have been limited to a heterologous system. In this study, we developed a genetic system for studying sRNAs in Chlamydia trachomatis and used it to identify four chlamydial sRNAs whose overexpression decreased the production of infectious bacteria. We also successfully utilized this genetic system to determine the target recognition sequence and mRNA targets of an uncharacterized, chlamydial sRNA named CtrR3. Overall, this work offers a generalizable approach for investigating the role of chlamydial sRNAs in their native organism.

Our group has found that protein S-glutathionylation serves as an important feedback inhibitor for superoxide (O2●-)/hydrogen peroxide (H2O2) production by several mitochondrial dehydrogenases. Since cytoplasmic oxidases can also serve as important reactive oxygen species (ROS) sources, we hypothesized that glutathionylation can also inhibit O2●-/H2O2 by these enzymes. We first focused our attention on using a purified xanthine oxidase (XO) of bacterial origin to discern if glutathionylation can shut down ROS production by this enzyme. Incubating XO in glutathione disulfide (GSSG) at a final concentration of 1 mM did not significantly alter ROS production. Additionally, incubating samples in up to 10 mM GSSG increased ROS production. However, diamide and disulfiram titrations in the presence of 1 mM GSH revealed that both glutathionylation catalysts were able to abolish O2●-/H2O2 by XO. Exposure of XO to glutaredoxin-1 (GRX1) and GSSG did not alter the rate of O2●-/H2O2 production. However, incubation with GSH and purified glutathione S-transferase (GST) almost abolished ROS production by XO. Similar results were collected with rat liver cytoplasm. Indeed, diamide and disulfiram significantly decreased ROS production by xanthine oxidoreductase (XOR). Additionally, incubating the cytoplasm in GSH and GST led to a significant decrease in XOR activity. Immunoblot analyses revealed that immunoreactive bands corresponding to XOR were glutathionylated by diamide. Collectively, our findings demonstrate for the first time that cytoplasmic ROS sources, such as XOR, can also be inhibited by glutathionylation and these reactions are enzymatically mediated by GST. Additionally, we found that bacterial XO is also a target for glutathionylation.

Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca2+ signaling. The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes.

Repetitive subconcussive head impact exposure has been associated with clinical and MRI changes in some non-concussed contact sport athletes over the course of a season. However, analysis of human tolerance for repeated head impacts is complicated by concussion and head impact exposure history, genetics, and other personal factors. Therefore, the objective of the current study was to develop a rodent model for repetitive subconcussive head impact exposure that can be used to understand injury mechanisms and tolerance in the human. This study incorporated the Medical College of Wisconsin Rotational Injury Model to expose rats to multiple low-level head accelerations per day over a 4-week period. The peak magnitude of head accelerations were scaled from our prior human studies of contact sport athletes and the number of exposures per day were based on the median (moderate exposure) and 95th percentile (high exposure) number of exposures per day across the human sample. Following the exposure protocol, rats were assessed for cognitive deficits, emotional changes, blood serum levels of axonal injury biomarkers, and histopathological evidence of injury. High exposure rats demonstrated cognitive deficits and evidence of anxiety-like behaviors relative to shams. Moderate exposure rats did not demonstrate either of those behaviors. Similarly, high exposure rats had histopathological evidence of gliosis [i.e., elevated Iba1 intensity and glial fibrillary acidic protein (GFAP) volume relative to shams] in the basolateral amygdala and other areas. Blood serum levels of neurofilament light (NFL) demonstrated a dose response relationship with increasing numbers of low-level head acceleration exposures with a higher week-to-week rate of NFL increase for the high exposure group compared to the moderate exposure group. These findings demonstrate a cumulative effect of repeated low-level head accelerations and provide a model that can be used in future studies to better understand mechanisms and tolerance for brain injury resulting from repeated low-level head accelerations, with scalable biomechanics between the rat and human.

Thoracic outlet syndrome (TOS) is a neurovascular condition involving the upper extremity, which is known to occur in individuals who perform chronic repetitive upper extremity activities. We prospectively evaluate the incidence of TOS in high-performance musicians who played bowed string musicians. Sixty-four high-performance string instrument musicians from orchestras and professional musical bands were included in the study. Fifty-two healthy volunteers formed an age-matched control group. Bilateral upper extremity duplex scanning for subclavian vessel compression was performed in all subjects. Provocative maneuvers including Elevated Arm Stress Test (EAST) and Upper Limb Tension Test (ULTT) were performed. Abnormal ultrasound finding is defined by greater than 50% subclavian vessel compression with arm abduction, diminished venous waveforms, or arterial photoplethysmography (PPG) tracing with arm abduction. Bowed string instruments performed by musicians in our study included violin (41%), viola (33%), and cello (27%). Positive EAST or ULTT test in the musician group and control group were 44%, and 3%, respectively (p = 0.03). Abnormal ultrasound scan with vascular compression was detected in 69% of musicians, in contrast to 15% of control subjects (p = 0.03). TOS is a common phenomenon among high-performance bowed string instrumentalists. Musicians who perform bowed string instruments should be aware of this condition and its associated musculoskeletal symptoms.