Pontine tubercular abscess: A rare presentation of CNS tuberculosis masquerading as glioma in a child.

Tuberculous brain abscess (TBA) in a child was initially misdiagnosed as glioma. Two craniotomies, abscess drainage, and anti-tubercular therapy led to recovery. Pontine TBA, though rare and atypical, can have better outcome with timely intervention.

Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2.

OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

Evaluating aortomesenteric parameters in a tertiary center of Nepal for superior mesenteric artery syndrome diagnosis and risk factors: cross-sectional study.

Background and objectives: This study examines aortomesenteric angle (AMA) and distance (AMD), which are critical in superior mesenteric artery (SMA) syndrome. Addressing the scarcity of SMA cases, the research explores potential links with lower BMI and aims to establish normative data for diagnostic and predictive purposes, using contrast-enhanced computed tomography (CT) scans across various BMI and sex categories. Methodology: A retrospective quantitative cross-sectional study was conducted on 189 patients undergoing abdominal contrast-enhanced CT scans between December 2019 and December 2020. Ethical clearance was obtained, and participants provided informed consent. Exclusion criteria targeted specific medical histories. Patient demographics, BMI categories, and imaging data were recorded. Helical 128-slice CT scans were employed, with sagittal-oblique multiplanar reconstructions for parameter assessments. Statistical analysis utilized SPSS 26.0, including Pearson correlation coefficients and mean calculations. Results: The study reveals a mean AMA of 54.07°±8.53° and a mean distance of 16.25±3.44 mm. Elevated BMI is found to positively correlate with AMA and distance, indicating that higher BMI values may augment these parameters, with an additional positive correlation observed between AMA and distance. No significant correlations are found with patient age or sex. Conclusion: The study concludes that decreased BMI may pose a potential risk for SMA syndrome, as evidenced by the observed correlations with aortomesenteric parameters. Understanding these normal values in the Nepalese population is critical for accurate diagnoses and predictions using CT scans. The research highlights the impact of demographic factors on these parameters and emphasizes their significance in clinical assessments related to SMA syndrome.

Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells.

Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021).

Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders.

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

Overcoming Barriers to Accessing Surgery and Rehabilitation in Low and Middle-Income Countries: An Innovative Model of Patient Navigation in Nepal.

BACKGROUND: Injury and disability are prominent public health concerns, globally and in the country of Nepal. Lack of locally available medical infrastructure, socioeconomic barriers, social marginalization, poor health literacy, and cultural barriers prevent patients from accessing surgical and rehabilitative care. Overcoming these barriers is an insurmountable challenge for the most vulnerable and marginalized, resulting in absence of treatment or even death. METHODS: Sundar Dhoka Saathi Sewa (SDSS), a non-government organization, provides a patient navigation service which facilitates referrals to tertiary centers from Nepal's most remote areas. Specific criteria ensure that patient referrals are appropriate in regard to clinical and socioeconomic need, while comprehensive counselling helps guide the patient and family. The SDSS staff meet patients upon arrival in Kathmandu and facilitate admission to the appropriate tertiary hospital. They advocate for the patient, provide medicine, supply food and cover all treatment costs. RESULTS: This project has enabled access to treatment for more than 1200 children for conditions leading to long-term disability and/or congenital heart disease. Interventions include a wide range of surgical and rehabilitative procedures such as complex orthopedics, cleft lip and palate, congenital talipes equinovarus, burn contractures, neurological cases, and cardiac surgery for valvular disease, septal defects and other congenital malformations. DISCUSSION: The SDSS model of patient navigation is effective in overcoming the barriers to access surgical care and rehabilitation in Nepal. The success is owed to committed international donors, capacity building, effective counselling, advocacy, compassion, and community. We believe that this model could be replicated in other LMICs.

Evaluation of Breast Mass by Mammography and Ultrasonography with Histopathological Correlation.

BACKGROUND: Mammography, ultrasound and Magnetic Resonance Imaging are the available modalities for the evaluation of breast masses. Advances and ongoing improvements in imaging technologies have improved the sensitivity of breast cancer detection and diagnosis, but each modality is most beneficial when utilized according to individual traits such as age, risk factors, and breast density. However, pathological diagnosis is most crucial for the treatment of breast masses. METHODS: A cross-sectional study were conducted from January 2017 to April 2018. There were total of 50 patients with clinically diagnosed palpable breast lumps who attended Gynaecological OPD/surgical OPD/medicine OPD in the study period. The patients above 30 years were evaluated by mammography and ultrasound in Department of Radiology, National Academy of Medical Sciences, Bir Hospital. The patients were then send for FNAC/biopsy and histopathology examination. Data were collected and analyzed using SPSS version 16. Specificity and sensitivity of MG and USG individually and in combination to determine the nature of breast lump in relation to histopathological findings were calculated. RESULTS: Ultrasound had 88.90% sensitivity and 68.80% specificity whereas mammogram had 94.40% and 87.50% sensitivity and specificity respectively. When combined, both sensitivity of diagnosing malignant lesions increases up to 94.4% and specificity decreases up to 31.2%. Most of the variables of ultrasound and mammography (except density of the lesion) had significance in predicting nature of the lesion (p< 0.05). CONCLUSIONS: Combined Mammography and Ultrasound had higher sensitivity than sensitivity rate observed for either single modality. A combined Mammography and Ultrasound approach to detect breast diseases was significantly more helpful in accurate evaluation of breast pathologies.

PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase.

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Selective Persulfide Detection Reveals Evolutionarily Conserved Antiaging Effects of S-Sulfhydration.

Life on Earth emerged in a hydrogen sulfide (H2S)-rich environment eons ago and with it protein persulfidation mediated by H2S evolved as a signaling mechanism. Protein persulfidation (S-sulfhydration) is a post-translational modification of reactive cysteine residues, which modulate protein structure and/or function. Persulfides are difficult to label and study due to their reactivity and similarity with cysteine. Here, we report a facile strategy for chemoselective persulfide bioconjugation using dimedone-based probes, to achieve highly selective, rapid, and robust persulfide labeling in biological samples with broad utility. Using this method, we show persulfidation is an evolutionarily conserved modification and waves of persulfidation are employed by cells to resolve sulfenylation and prevent irreversible cysteine overoxidation preserving protein function. We report an age-associated decline in persulfidation that is conserved across evolutionary boundaries. Accordingly, dietary or pharmacological interventions to increase persulfidation associate with increased longevity and improved capacity to cope with stress stimuli.

Measurement of Protein Persulfidation: Improved Tag-Switch Method.

Hydrogen sulfide (H2S) is an endogenously produced signaling gasotransmitter, generated by the enzymes cystathionine γ-lyase, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase. The involvement of H2S in numerous physiological, as well as pathophysiological conditions, was established over the past decade. However, the exact mechanism(s) of regulation of the biological functions by H2S are under active investigations. It is proposed that the oxidative posttranslational modification of protein cysteine residues, known as persulfidation, could be the main mechanism of action of H2S. Protein persulfides show similar reactivity to thiols, which represents one of the main obstacles in the development of a reliable method for detection of this specific protein modification. Subsequently, having a selective method for persulfide detection is of utmost importance in order to fully understand the physiological and pathophysiological role of H2S. Several methods have been proposed for the detection of protein persulfidation, all of which are highlighted in this chapter. Furthermore, we provide a detailed description and protocol for the first selective persulfide labeling method, a tag-switch method, developed in our group.

MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

Cytochrome c Reduction by H2S Potentiates Sulfide Signaling.

Hydrogen sulfide (H2S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by H2S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H2S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H2S oxidized. H2S increases O2 consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H2S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.