Flash VEP in Clinically Stable Pre-Term and Full-Term Infants

Purpose Pre-term infants are at risk of abnormal visual development that can range from subtle to severe. The aim of this study was to compare flash VEPs in clinically stable pre-term and full-term infants at 6 months of age. Methods Twenty-five pre-term and 25 full-term infants underwent flash VEP testing at the age of 6 months. Monocular VEPs were recorded using flash goggles on a RETIscan system under normal sleeping conditions. Amplitude and peak time responses of the P2 component in the two eyes were averaged and compared between the two groups. Multiple regression analyses were performed to assess the relationship of the P2 responses with birth weight (BW) and gestational age (GA). Results At 6 months corrected age, pre-term infants had significantly delayed P2 peak times than full-term infants (mean difference: 10.88 [95% CI 4.00–17.76] ms, p = 0.005). Pre-term infants also showed significantly reduced P2 amplitudes as compared to full-term infants (mean difference: 2.36 [0.83–3.89] µV, p = 0.003). Although the regression model with GA and BW as fixed factors explained 20% of the variance in the P2 peak time (F2,47 = 5.98, p = .0045), only GA showed a significant negative relationship (β = −2.66, p = .003). Neither GA (β = 0.21, p = .28) nor BW (β = 0.001, p = .32) showed any relationship with P2 amplitude. Conclusions Our results demonstrate that, compared with full-term infants, clinically stable pre-term infants exhibit abnormal flash VEPs, with a delay in P2 peak time and a reduction in P2 amplitude. These findings support a potential dysfunction of the visual pathway in clinically stable pre-term infants as compared to full-term infants

Prescribing patterns in patients with chronic liver and kidney disease in a tertiary care hospital

Background: Liver diseases are major cause of mortality and morbidity worldwide. It is the 12th leading cause of death liver diseases can be classified as acute if the onset of symptom does not exceed six months or chronic if symptoms persist beyond this period. According to the recently available World Health Organization. The aim of study is to facilitate rational use of medicines. Methods: This study is a prospective, observational, single center study which include patients aged ≥18 years, diagnosis of liver diseases with or without co-morbidities and is conducted at out-patient of Medicine department, Rajindra Medical College and Hospital, Patiala. Results: In this study, total of 97 prescriptions of patients with liver disease were analyzed. Out of 97 patients, the majority of patients were male. In ALD, males were 32 (78%) whereas female were 9 (22%) while in CLD males were 47 (84%) and females were 9 (16%).While observing the LFT profile of patients with ALD common tests were observed which includes total bilirubin (1.82±2.42), SGOT (96.81±117.49) and SGPT (94.78±142.94) and in patients with CLD common tests were observed which includes total bilirubin (2.50±3.63), SGOT (67.50±43.04), SGPT (47.10±33.12), blood urea (46.92±24.14) and alkaline phosphatase (147.02±63.14). Conclusions: The study interprets the prescribing pattern of drugs used in patients with ALD and CLD and observed that vitamins and minerals and antibiotics were the most prescribed in order to avoid further complications followed by hepatoprotective agents, antiulcer drugs, antihypertensives and laxatives.

Refractive Status in Nepalese Pre-Term and Full-Term Infants Early in Life.

SIGNIFICANCE: This study suggests that pre-term infants, even without retinopathy of prematurity, are at risk for abnormal refractive development and informs the need for close monitoring of refractive error in such infants, regardless of their retinopathy of prematurity status. PURPOSE: The present study aims to investigate the refractive error trend in Nepalese pre-term infants without retinopathy of prematurity (ROP) in the first 6 months of life and explore the association of refractive error with birth weight (BW) and gestational age (GA). METHODS: Thirty-six pre-term infants without ROP and 40 full-term infants underwent cycloplegic retinoscopy at birth, term (for pre-term only), 3 months, and 6 months chronologically. Refractive status was classified into emmetropia (mean spherical equivalent refraction [SER] 0 to +3.00D), myopia (SER +3.00D). Refractive parameters at various age points were compared between the pre-term and full-term infants using general linear model repeated measures ANOVA. RESULTS: At birth, the SER in the pre-term infants was +0.84 ± 1.72D; however, there was a shift toward myopia at 6 months of age (SER = -0.33 ± 1.95D). There was a significant difference in SER, astigmatism, and anisometropia between pre-term and full-term infants by 6 months of age (P < .01). Astigmatism and anisometropia showed an increasing trend with age in pre-term infants (P < .05 at 6 months) in contrast to a decreasing trend in full-term infants (P < 0.05 at 3 and 6 months). In pre-term infants, there was a statistically significant positive relationship between GA and SER (β = 0.32, R = 17.6%, P < .05) but a negative relationship between BW and astigmatism (β = -1.25, R = 20.6%, P < .01). CONCLUSIONS: Pre-term infants who do not develop ROP show a trend toward increasing myopia and demonstrate greater astigmatism and anisometropia than full-term infants in their first 6 months of life

Deep-learning assisted detection and quantification of (oo)cysts of Giardia and Cryptosporidium on smartphone microscopy images

The consumption of microbial-contaminated food and water is responsible for the deaths of millions of people annually. Smartphone-based microscopy systems are portable, low-cost, and more accessible alternatives for the detection of Giardia and Cryptosporidium than traditional brightfield microscopes. However, the images from smartphone microscopes are noisier and require manual cyst identification by trained technicians, usually unavailable in resource-limited settings. Automatic detection of (oo)cysts using deep-learning-based object detection could offer a solution for this limitation. We evaluate the performance of three state-of-the-art object detectors to detect (oo)cysts of Giardia and Cryptosporidium on a custom dataset that includes both smartphone and brightfield microscopic images from vegetable samples. Faster RCNN, RetinaNet, and you only look once (YOLOv8s) deep-learning models were employed to explore their efficacy and limitations. Our results show that while the deep-learning models perform better with the brightfield microscopy image dataset than the smartphone microscopy image dataset, the smartphone microscopy predictions are still comparable to the prediction performance of non-experts.Comment: 18 pages (including supplementary information), 4 figures, 7 tables, submitting to Journal of Machine Learning for Biomedical Imagin

Granulovirus PK-1 kinase activity relies on a side-to-side dimerization mode centered on the regulatory αC helix

The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases

Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys290) that lies adjacent to Thr288, a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

ONE SENTENCE SUMMARY A Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2 in vitro and lead to rapid on-target degradation of TRIB2 in human cancer cells. SHORT ABSTRACT Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the AKT signaling module. Substantial evidence demonstrates that TRIB2 dysregulation is important in multiple human tumors. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine rich region and interacts with a peptide motif in its own C-terminal tail. We demonstrate that TRIB2 is a target for previously described small molecule protein kinase ‘inhibitors’, which were originally designed to inhibit the catalytic domain of EGFR/HER2 tyrosine kinases. Using thermal-shift assays and drug repurposing, we classify ligands that stabilize or destabilize the TRIB2 pseudokinase domain. TRIB2 destabilizing agents, including the clinical inhibitor afatinib, lead to rapid and on-target TRIB2 protein degradation in tumor cells, eliciting tractable effects on cell signaling and survival. Our data identifies leads for further development of TRIB2-degrading drugs and highlights compound-induced TRIB2 downregulation, which might be mechanistically relevant for other catalytically-deficient (pseudo)kinases targeted by small molecules. FULL ABSTRACT A major challenge associated with biochemical and cellular analysis of pseudokinases is the lack of target-validated small molecule ligands with which to probe molecular function. Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, which includes the canonical AKT signaling module. There is substantial evidence that human TRIB2 is a therapeutic target in both solid tumors and blood cancers. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine-rich region and interacts with a peptide motif in its own C-terminal tail, which was previously shown to drive interaction with cellular E3 ubiquitin ligases. In this study we demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the canonical catalytic domain of the tyrosine kinases EGFR/HER2. Using a thermal-shift assay, we discovered TRIB2 ligands within the Published Kinase Inhibitor Set (PKIS), and employed a drug repurposing approach to classify compounds that either stabilize or destabilize TRIB2 in vitro . Remarkably, TRIB2 destabilizing agents, including the clinical covalent drug afatinib, lead to rapid and on-target TRIB2 degradation in human cells, eliciting tractable effects on signaling and survival. Our data reveal the first drug-leads for development of TRIB2-degrading ligands, which will also be invaluable for unravelling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein downregulation through drug ‘off-targets’ might be relevant for other inhibitors that serendipitously target pseudokinases. ABBREVIATIONS DSF Differential Scanning Fluorimetry EGFR Epidermal Growth Factor Receptor HER2 Human Epidermal Growth Factor Receptor 2 MS Mass spectrometry MST MicroScale Thermophoresis PKIS Published Kinase Inhibitors Set TRIB2 Tribbles 2 TSA Thermal Stability Assa

Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases

Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The doublecortin-like kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory ‘tail’ segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations, and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to ‘supercharge’ the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other calcium calmodulin kinases (CAMKs), and a ‘Swiss Army’ assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for autoregulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically divergent DCLK1 modulators, stabilizers, or degraders.</jats:p

Discovery of a Cushing’s syndrome protein kinase A mutant that biases signaling through type I AKAPs

Adrenal Cushing’s syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing’s driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAc W196G is unexpectedly distinct from other described Cushing’s variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653–cubic angstrom cleft between the catalytic core of PKAc W196G and type II regulatory subunits (RII), but only a 395–cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAc W196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing’s syndrome. </jats:p