Longitudinal single cell atlas identifies complex temporal relationship between type I interferon response and COVID-19 severity.

Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia.

Primary resistance to tyrosine kinase inhibitors (TKIs) is a significant barrier to optimal outcomes in chronic myeloid leukemia (CML), but factors contributing to response heterogeneity remain unclear. Using single-cell RNA (scRNA) sequencing, we identified 8 statistically significant features in pretreatment bone marrow, which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis [BC] transformation). Employing machine-learning, we identified leukemic stem cell (LSC) and natural killer (NK) cell gene expression profiles predicting imatinib response with >80% accuracy, including no false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor [ERP] expansion in vivo (P < .05), and a 2- to 10-fold (6.3-fold in group A vs 1.09-fold in group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared with myeloid progenitors (P < .001). These LSC features were lost with progressive resistance, and MYC- and IRF1-driven inflammatory regulons were evident in patients who progressed to transformation. Patients with MMR also exhibited a 56-fold expansion (P < .01) of a normally rare subset of hyperfunctional adaptive-like NK cells, which diminished with progressive resistance, whereas patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance. Finally, we developed antibody panels to validate our scRNA-seq findings. These panels may be useful for prospective studies of primary resistance, and in assessing the contribution of predetermined vs acquired factors in TKI response heterogeneity.

Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer.

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).

Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response.

The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days postinfection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-α) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-2, implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients.

RCA2: a scalable supervised clustering algorithm that reduces batch effects in scRNA-seq data.

The transcriptomic diversity of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Unsupervised clustering of SC transcriptomes, which is the default technique for defining cell types, is prone to group cells by technical, rather than biological, variation. Compared to de-novo (unsupervised) clustering, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects and data quality artifacts. Here, we present RCA2, the first algorithm to combine reference projection (batch effect robustness) with graph-based clustering (scalability). In addition, RCA2 provides a user-friendly framework incorporating multiple commonly used downstream analysis modules. RCA2 also provides new reference panels for human and mouse and supports generation of custom panels. Furthermore, RCA2 facilitates cell type-specific QC, which is essential for accurate clustering of data from heterogeneous tissues. We demonstrate the advantages of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Scalable supervised clustering methods such as RCA2 will facilitate unified analysis of cohort-scale SC datasets.

Long-term complications of stroke and secondary prevention: an overview for primary care physicians.

Despite a decline in mortality from stroke, the annual incidence in the general population is increasing. For many stroke survivors and their families, the acute stroke is the beginning of an ongoing struggle with physical impairment and subsequent disability. Over time, the immediate clinical consequences of the stroke are complicated by a variety of lesser-known medical, musculoskeletal and psychosocial difficulties. The primary care physician is best positioned to optimise chronic disease control, reduce risk and manage complications of stroke. Early screening and appropriate management is key. Instituting secondary prevention and attention to bowel and bladder problems can help reduce medical complications and re-admissions, while adequate analgesia, positioning/splinting of limbs and physiotherapy can lessen discomfort and preventable suffering. Primary care physicians can identify and treat post-stroke mood issues and involve psychological counselling for patients and caregivers. Adequate education and support may restore the independence of patients with stroke or minimise any resultant dependency.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata).

In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.

A terpenoid phytoalexin plays a role in basal defense of Nicotiana benthamiana against Potato virus X.

Terpenoid phytoalexins function as defense compound against a broad spectrum of pathogens and pests in the plant kingdom. However, the role of phytoalexin in antiviral defense is still elusive. In this study, we identified the biosynthesis pathway of a sesquiterpenoid phytoalexin, capsidiol 3-acetate as an antiviral response against RNA virus Potato Virus X (PVX) in Nicotiana benthamiana. NbTPS1 and NbEAH genes were found strongly induced by PVX-infection. Enzymatic activity and genetic evidence indicated that both genes were involved in the PVX-induced biosynthesis of capsidiol 3-acetate. NbTPS1- or NbEAH-silenced plant was more susceptible to PVX. The accumulation of capsidiol 3-acetate in PVX-infected plant was partially regulated by jasmonic acid signaling receptor COI1. These findings provide an insight into a novel mechanism of how plant uses the basal arsenal machinery to mount a fight against virus attack even in susceptible species.

Results of urinary dissolution therapy for radiolucent calculi.

PURPOSE: In this paper we present our experience with dissolution therapy of radiolucent calculi. MATERIALS AND METHODS: This was a retrospective analysis of patients who were offered urinary dissolution therapy between January 2010 and June 2011. Patients were treated with tablets containing potassium citrate and magnesium oxide. Partial dissolution was defined as at least a 50% reduction in stone size. Patients with complete or partial dissolution were classified in the successful dissolution group. Patients with no change, inadequate reduction, increase in stone size and those unable to tolerate alkali therapy were classified as failures. Patient sex, stenting before alkalinization, stone size, urine pH at presentation and serum uric acid levels were analyzed using Fisher t-test for an association with successful dissolution. RESULTS: Out of 67, 48 patients reported for follow up. 10 (15%) had complete dissolution and 13 (19%) had partial dissolution. Alkalinization was unsuccessful in achieving dissolution in 25 (37%). Stenting before alkalinization, patient weight (< 60 vs. > 75kg) and serum uric acid levels (≤ 6 vs. > 6) were the only factors to significantly affected dissolution rates (p = 0.039, p 0.035, p 0.01 respectively). CONCLUSIONS: A policy of offering dissolution therapy to patients with radiolucent calculi had a successful outcome in 34% of patients.

Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants.

Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.

Clinical and radiological features of pediatric cerebellar anaplastic oligodendrogliomas.

Oligodendrogliomas constitute 4-7% of primary intracranial gliomas in adult and 1% of primary central nervous system tumours in children. The authors report two unusual pediatric cases of anaplastic oligodendroglioma occurring in the cerebellum. The patient with a well-circumscribed lesion with 1p/19q co-deletion had a favorable outcome in comparison with the patient with a diffuse lesion and intact 1p/19q. The discussion elucidates the surgical outcome of such pediatric cerebellar anaplastic oligodendrogliomas in relation to their radiological findings.

Isolated sellar tuberculoma presenting with panhypopituitarism: clinical, diagnostic considerations and literature review.

Tuberculosis, a common cause for chronic intracranial infections can mimic varied intracranial pathologies including tumours. Pituitary tuberculomas are rare lesions and are often diagnosed pre-operatively as pituitary tumours. We report a case of a 31-year-old lady with a sellar-suprasellar lesion who presented with panhypopituitarism. The patient underwent a trans-nasal, trans-sphenoidal surgical decompression of the lesion. Histopathology revealed a tuberculous lesion in the pituitary. The characteristic radiological features of sellar tuberculomas are discussed along with a review of literature. Atypical sellar radiology in the presence of a thickened pituitary stalk could point to pathology other than pituitary adenoma, possibly a chronic inflammatory condition like tuberculoma.

Invasive sphenocavernous aspergilloma complicating an operated case of acromegaly.

There is a surge in fungal infections of the central nervous system due to an increase in the incidence of immunocompromised state and widespread use of antimicrobials. Despite myriad clinical presentations and skull base syndromes associated with Aspergillus infections, aspergilloma in the sella is relatively rare. We report a rare case of aspergilloma of the sella and parasellar region in a patient operated for acromegaly. A 30-year-old diabetic patient underwent two surgeries for a growth hormone-secreting pituitary adenoma, which included a transsphenoidal approach. During his last admission, magnetic resonance imaging revealed a heterogeneous mass in the sella, suprasellar region, sphenoid sinus, and cavernous sinus with multiple areas of necrosis. His presentation, surgical treatment, and response to antifungal therapy are discussed. The role of prior surgery and related comorbidities in developing the invasive granuloma are highlighted.

Coexisting intracranial tumors with pituitary adenomas: genetic association or coincidence?

The co-occurrence of two or more brain tumors with different histological features is rare. The authors report three rare cases of intracranial tumors associated with pituitary adenomas. Two of the pituitary tumors were functioning adenomas: a prolactinoma and a thyrotropin secreting adenoma. Two of the associated intracranial neoplasms were gliomas and one was a meningioma. Radiological and clinical examination for syndromal association was negative in all cases. We briefly discuss the presentation and treatment options of these cases and review the 19 previous publications in the literature of pituitary tumors occurring in association with other neoplasms and explore the possible links underlying these co-occurring neoplasms. Our three cases represent 0.86% of all pituitary tumors operated at our institute over a 9-year period.

Paradoxical embolus across atrial septal defect and posterior circulation infarct in neurosurgical patients.

Paradoxical embolism through a patent foramen ovale or atrial septal defect is increasingly recognized in association with embolic strokes, with the advent of modern echocardiography. The authors describe two neurosurgical cases with such an anomaly that suffered nonfatal embolic stroke in the cerebellum. The patients developed posterior inferior cerebellar artery infarcts, which were managed conservatively and had no neurological deficits. They evaluate possible factors for emboli to occur in a perioperative neurosurgical setting and provide a brief review of literature on preoperative management of patients with this cardiac defect. Closure of cardiac shunts is warranted before non-emergent neurosurgical operations. During emergency craniotomies, proper precautions should be taken to reduce the incidence of venous air embolism and paradoxical embolism.

Chronic brain abscess secondary to a retained wooden foreign body: diagnostic and management dilemmas.

A four-year-old child presented with recent onset generalized tonic clonic seizures. She was operated for a suspected intracranial tuberculoma and was found to harbor an intra-parenchymal retained wooden foreign body with a chronic abscess. The foreign body had entered the brain parenchyma after a minor head injury, sustained three years earlier. She was asymptomatic for the intervening three years. The initial diagnosis was missed by several physicians. A retained wooden fragment via a transtemporal apparently closed head injury is an extremely rare event. The present study reveals the diagnostic and therapeutic challenges and stresses the importance of high degree of suspicion to diagnose retained intracranial foreign bodies and the need for early surgical exploration, to avoid chronic and potentially life threatening neurological complications.

Diagnostic and clinical implications of pituicytoma.

Pituicytoma is a rare, indolent, benign tumor of the sellar and suprasellar region arising from pituicytes of the neurohypophysis. It is most often diagnosed pre-operatively as a pituitary adenoma. We report two patients with pituicytoma operated on at our Institute over 8 years. Imaging of one patient showed a parasellar and medial temporal extension, which has not been reported to our knowledge. The radiological features that help distinguish this low-grade tumor from other sellar and parasellar tumors are discussed along with its distinct histological findings. The authors also review the literature on its clinical presentation, diagnosis, surgical management and outcome.

Neurological complications and surgical outcome in patients with anterior choroidal segment aneurysms.

INTRODUCTION: Choroidal segment aneurysms occur in close relation to terminal internal carotid branches and concomitant anatomical variations in the anterior choroidal artery. The authors evaluate the demographics, presentation, operative techniques, and surgical outcome in 13 patients with this aneurysm. METHODS: After identifying the study group from a retrospective review of all supraclinoid ICA aneurysms operated in our institution over an 8-year period, analysis was done with respect to the radiological and operative findings and clinical follow-up. Statistical significance for outcome was analyzed using two-tailed Fisher's exact test (p < 0.05). RESULTS: The incidence of choroidal segment aneurysm was 3.02%. Six (46%) patients had Fisher grade III bleed on imaging. Nine (69%) patients had a good surgical outcome. Three patients had ischemic stroke in the anterior choroidal territory. Patient demographics, Hunt and Hess score at presentation, Fisher score on computed tomogram scan, aneurysm characteristics and clip configuration had no bearing on surgical outcome. CONCLUSIONS: In patients with anterior choroidal artery aneurysms managed surgically, meticulous surgical exposure, identification of anatomical variations of parent artery, appropriate clip selection and usage of intra-operative Doppler limit ischemic complications, and allay the development of the morbid clinical syndrome associated with anterior choroidal territory infarction.