SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status.

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

Association of Serum Iron Studies in COVID Associated Mucormycosis with Stage of the Disease.

Mucormycosis (Zygomycosis) is a rare and lethal invasive fungal infection, often acute and extremely severe caused by opportunist and ubiquitous fungi belonging to the class Phygomycetes, subclass Zygomycetes, order Mucorales, family Mucoraceae. India has reported surge in cases of post COVID 19 Mucormycosis over the past few months due to the increasing frequency of risk factors like corticosteroid therapy, uncontrolled diabetes, DKA, neutropenia and iron overload. Patients with a history of COVID-19 infection are at increased risk of developing fungal infections like Mucormycosis. The emergence of COVID-19 associated mucormycosis (CAM) across several nations, particularly India, warrants a detailed study to identify potential contributing factors. MATERIAL: This cross sectional study conducted at Bowring and Lady Curzon Hospital, Bangalore, involving 75 subjects diagnosed with CAM either clinically, radiological or microbiologically. The objective was to study the clinical profile of patients with COVID associated Mucormycosis and to correlate the levels of Serum ferritin and iron profile with severity and extent of disease in COVID associated Mucormycosis patients Data was collected on demographic details, co morbidities, vaccination status, history of treatment with remedesvir, oxygen therapy or steroid use, complications of past COVID 19 infection and stage of current Mucormycosis infection. Clinical outcome of the patients measured based on Iron profile, length of hospital stay, need for ICU admission, presence of diabetic ketoacidosis and mortality. The blood investigations which included were CBC with differential leukocyte count, qCRP, FBS, PPBS, HbA1c serum iron studies and serum ferritin. OBSERVATION: The mean age of the subjects was 48.19 with 52 males, 23 females. Among 75 patients with CAM, 90.7% were unvaccinated against COVID-19, 62.7% had oxygen usage and steroid therapy, 44% had use of remedesvir. Most common co morbidity was diabetes mellitus 60% with 20% of patients having DKA. Rhino orbital-cerebral mucormycosis(Stage 4- 44.6%) was the most common clinical presentation. The mean serum iron (50.37) and TIBC (255.37) were significantly higher in Stage 4 CAM cases compared with less invasive stage 2 CAM cases. Patients with Stage 4 CAM had elevated levels of inflammatory markers LDH (292) DDimer (457) CRP(74.64). Case fatality rates of CAM was 12%. CONCLUSION: The results of this study revealed significant correlation between the clinical severity of CAM and higher mortality, increased serum iron levels and inflammatory markers in this population of patients. Therefore, patients with elevated levels of available serum iron are uniquely susceptible to mucormycosis infection, suggesting dysregulated iron metabolism in its pathogenesis.

STEMI in Young Befogged by Aluminum Phosphide Toxicity-Role of ECMO as Salvage Therapy and Trimetazidine and Magnesium to Suppress Arrhythmias.

INTRODUCTION: Aluminum phosphide poisoning (ALP) has a high-mortality rate despite intensive care management, primarily because it causes severe myocardial depression. This case report highlights the subset of ALP patients presenting as ST elevation myocardial infarction (STEMI) with profound myocardial dysfunction and multiorgan failure and successfully treated with extracorporeal membrane oxygenation (ECMO), trimetazidine, and magnesium. CASE DESCRIPTION: A 25-year-old man without any comorbidities was brought to emergency department with dyspnea and hypotension. His electrocardiograph (ECG) revealed STEMI with elevated troponin levels, arterial blood gas (ABG) showed severe metabolic acidosis, and echocardiography (echo) revealed ejection fraction 15%. He was initiated on venoarterial (VA) ECMO in view of refractory hypotension. History of consumption of three tabs of celphos was revealed later by the family members. He progressed to cardiogenic shock, arrhythmias, respiratory failure, acute kidney injury with severe lactic acidosis, liver injury, pancreatitis, and disseminated intravascular coagulation (DIC). He was successfully supported by ECMO, hemodialysis, magnesium, trimetazidine, N-acetyl cysteine, inotropes, and blood products. He was weaned off ECMO on day 6 and was discharged home on day 12. Despite his severe and confounding clinical presentation, he had complete normalization of end-organ dysfunction with no neurological sequela. This case demonstrates the high index of suspicion required for ALP, given the potential for rapid progression and severe multiorgan toxicity. This report also highlights the importance of early referral to a tertiary care center with ECMO capability and also the role of magnesium and trimetazidine to suppress arrhythmias. CONCLUSION: Aluminum phosphide poisoning can present as STEMI with cardiogenic shock resulting in acute kidney injury, liver injury, pancreatitis, and DIC. Venoarterial ECMO provides an effective means of support until the recovery of organ function. Trimetazidine and magnesium are helpful in suppressing fatal arrhythmias. This report emphasizes that early recognition and early institution of ECMO can save many young lives who succumb to toxic effects of this poison. HOW TO CITE THIS ARTICLE: Rao CC, Himaaldev GJ. STEMI in Young Befogged by Aluminum Phosphide Toxicity-Role of ECMO as Salvage Therapy and Trimetazidine and Magnesium to Suppress Arrhythmias. Indian J Crit Care Med 2020;24(8):727-730.

PTAP motif duplication in the p6 Gag protein confers a replication advantage on HIV-1 subtype C.

HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro-Thr/Ser-Ala-Pro (PTAP) motif necessary for viral packaging. However, the biological significance of a duplication of the PTAP motif for HIV-1 replication and pathogenesis has not been experimentally validated. In a longitudinal study of two different clinical cohorts of select HIV-1 seropositive, drug-naive individuals from India, we found that 8 of 50 of these individuals harbored a mixed infection of viral strains discordant for the PTAP duplication. Conventional and next-generation sequencing of six primary viral quasispecies at multiple time points disclosed that in a mixed infection, the viral strains containing the PTAP duplication dominated the infection. The dominance of the double-PTAP viral strains over a genetically similar single-PTAP viral clone was confirmed in viral proliferation and pairwise competition assays. Of note, in the proximity ligation assay, double-PTAP Gag proteins exhibited a significantly enhanced interaction with the host protein tumor susceptibility gene 101 (Tsg101). Moreover, Tsg101 overexpression resulted in a biphasic effect on HIV-1C proliferation, an enhanced effect at low concentration and an inhibitory effect only at higher concentrations, unlike a uniformly inhibitory effect on subtype B strains. In summary, our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the Tsg101 host protein with a higher affinity. Our results have implications for HIV-1 pathogenesis, especially of HIV-1C.

Geographical distribution of primary & secondary dengue cases in India - 2017: A cross-sectional multicentric study.

Background & objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India. Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases. Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups. Interpretation & conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Outcome of Patients Admitted to a Tertiary Referral Intensive Care Unit with Urosepsis Needing Source Control.

INTRODUCTION: Urosepsis is one of the common causes of admission to the Intensive Care Unit (ICU). It has traditionally been treated with antibiotics, but surgical management with Double J [DJ] ureteral stents is gaining popularity. This study compares patients with complicated urosepsis who underwent surgical source control by ureteral stenting with those managed medically. MATERIALS AND METHODS: The study enrolled patients admitted to a tertiary adult ICU with a diagnosis of urosepsis over a period of 2 years. The primary outcomes were renal replacement therapy (RRT) requirement and ICU mortality. The secondary outcomes were ICU and hospital length of stay, ventilator-free days, and inotrope free days. Patients were divided those with obstructive and nonobstructive urinary tract infection (UTI). RESULTS: A total of 58 patients met the criteria, of who 32 had obstructive UTI and were included in Group A, with the remaining 26 with nonobstructive UTI comprised Group B. In Group A, 27 patients underwent source control with ureteral DJ stenting, three patients recovered with medical management, and two who were advised source control did not consent to the procedure. Seventeen patients in Group A and seven patients in Group B required RRT (P = 0.044). There was no significant difference in ICU mortality, hospital mortality, and 28 days survival between the two groups. CONCLUSION: With early source control, obstructive UTI outcomes were comparable to nonobstructive UTI. However, despite undergoing ureteric stenting, more patients with obstructive UTI required RRT than those with nonobstructive UTI.

KSR1 regulates small-cell lung carcinoma tumor initiation and cisplatin resistance.

Small-cell lung cancer (SCLC) is designated a recalcitrant cancer due to its five-year relative survival rate of less than 7%. First line SCLC treatment has changed modestly in the last 40 years. The NeuroD1 subtype of SCLC (SCLC-N) commonly harbors MYC amplifications and other hallmarks of aggressive behavior. Finding novel therapeutic options that effectively eliminate residual disease observed after initial response to therapy is essential to improving SCLC patient outcome. Here we show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK signaling cascade is critical for clonogenicity and tumor initiation in vitro and in vivo in the highly aggressive, metastatic and therapy resistant NeuroD1 subtype of SCLC. Tumor-initiating cells (TICs) are reported as the sanctuary population within the bulk tumor responsible for therapeutic resistance and relapse. Previous studies concluded ERK activation was inhibitory to growth and tumor development. We show that signaling through KSR1 is conserved in SCLC-N and that it regulates tumor initiation through interaction with ERK. We further show that KSR1 mediates cisplatin resistance in SCLC-N cells. While 50% of control SCLC-N cells show resistance after 6 weeks of exposure to cisplatin, CRISPR/Cas9-mediated KSR1 knockout prevents resistance in >90% of SCLC-N cells. KSR1 KO also significantly enhances the ability of cisplatin to decrease SCLC-N TICs, indicating that targeting KSR1 might be selectively toxic to cells responsible for therapeutic resistance and tumor initiation. Thus, KSR1 function in SCLC-N serves as a novel model for understanding the role of KSR1-dependent signaling in normal and malignant tissues. These findings shed light on a key distinct protein responsible for regulation in SCLC-N tumors, and a potential subtype specific therapeutic target.

Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes.

Aims/hypothesis: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. Methods: Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls. Results: PD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN-α or IFN-□ treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretation: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death. Research in context: What is already known about this subject? (maximum of 3 bullet points): Extracellular vesicles (EVs) serve as paracrine effectors in the islet microenvironment in health and disease.Interferon-alpha (IFN-α) and IFN-gamma (IFN-□) are key cytokines contributing to type 1 diabetes pathophysiology and islet IFN signalling increases beta cell programmed death-ligand 1 (PD-L1) expression.Up-regulation of beta cell PD-L1 in the non-obese diabetic (NOD) mouse model delays progression of type 1 diabetes.What is the key question? (one bullet point only; formatted as a question): Do beta cells exposed to IFNs upregulate EV PD-L1 and can these changes be detected in circulation?What are the new findings? (maximum of 3 bullet points): IFN-α or IFN-□ exposure increases beta cell EV PD-L1 cargo in beta cell lines and human islets.PD-L1 is present on the surface of beta cell EVs, binds PD-1 and EV PD-L1 inhibits proliferation, activation and cytotoxicity of murine CD8 T cells.EV PD-L1 levels are higher in islet autoantibody positive individuals compared to nondiabetic controls and levels of circulating EV PD-L1 positively correlate with residual beta cell function in islet autoantibody positive individuals as well as in individuals with recent-onset type 1 diabetes.How might this impact on clinical practice in the foreseeable future? (one bullet point only): A beneficial effect of PD-L1+ EVs could ultimately be harnessed as an intervention to prevent autoimmune beta cell destruction. Circulating EV PD-L1 cargo has potential as a minimally invasive and informative biomarker to offer insights into the pathogenesis and progression of type 1 diabetes.

Association of digital measures and self-reported fatigue: a remote observational study in healthy participants and participants with chronic inflammatory rheumatic disease.

Background: Fatigue is a subjective, complex and multi-faceted phenomenon, commonly experienced as tiredness. However, pathological fatigue is a major debilitating symptom associated with overwhelming feelings of physical and mental exhaustion. It is a well-recognized manifestation in chronic inflammatory rheumatic diseases, such as Sjögren's Syndrome and Systemic Lupus Erythematosus and an important predictor of patient's health-related quality of life (HRQoL). Patient reported outcome questions are the key instruments to assess fatigue. To date, there is no consensus about reliable quantitative assessments of fatigue. Method: Observational data for a period of one month were collected from 296 participants in the United States. Data comprised continuous multimodal digital data from Fitbit, including heart rate, physical activity and sleep features, and app-based daily and weekly questions covering various HRQoL factors including pain, mood, general physical activity and fatigue. Descriptive statistics and hierarchical clustering of digital data were used to describe behavioural phenotypes. Gradient boosting classifiers were trained to classify participant-reported weekly fatigue and daily tiredness from multi-sensor and other participant-reported data, and extract a set of key predictive features. Results: Cluster analysis of Fitbit parameters highlighted multiple digital phenotypes, including sleep-affected, fatigued and healthy phenotypes. Features from participant-reported data and Fitbit data both contributed as key predictive features of weekly physical and mental fatigue and daily tiredness. Participant answers to pain and depressed mood-related daily questions contributed the most as top features for predicting physical and mental fatigue, respectively. To classify daily tiredness, participant answers to questions on pain, mood and ability to perform daily activities contributed the most. Features related to daily resting heart rate and step counts and bouts were overall the most important Fitbit features for the classification models. Conclusion: These results demonstrate that multimodal digital data can be used to quantitatively and more frequently augment pathological and non-pathological participant-reported fatigue.

The case of the neglected alphasyllabary: orthographic processing in Devanagari.

We applaud Ram Frost for highlighting the need for multicultural perspectives while developing universal models of visual word recognition. We second Frost's proposal that factors like lexical morphology should be incorporated besides purely orthographic features in modeling word recognition. In support, we provide fresh evidence from Hindi (written in Devanagari), an example of hitherto under-represented alphasyllabic orthographies, in which flexible encoding of aksara (character) position is constrained by the morphological structure of words.