Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

Comparison of SARS-CoV-2 indirect and direct RT-qPCR detection methods.

BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.

Accurate preoperative diagnosis of a Rathke cleft cyst with the aid of a novel classification for sellar cystic lesions and a diagnostic algorithm decision: Tools for differentiating cystic sellar lesions with a representative case.

Background: Rathke's cleft cyst (RCC) is a benign lesion in the sellar and suprasellar compartments. Similarly, pituitary adenomas can present with cystic morphology, making it a differential diagnosis when evaluating a patient with a cystic lesion in the sellar region. Surgical goals differ between RCCs and pituitary adenomas as the first can achieve remission of symptoms with cyst decompression in contrast to pituitary adenomas where complete resection would be the main goal. Imaging analysis alone may not be sufficient to define a preoperative surgical plan. The combination of imaging and conjoined use of validated tools may provide valuable insights to the clinician when defining a surgical approach. Case Description: We present a case of a 27-year-old male with a 3-month history of visual disturbances and headaches. Magnetic resonance imaging showed a cystic lesion in the sellar compartment with compression of nearby structures. The authors were able to accurately diagnose this sellar lesion as an RCC with the conjoined aid of two classifications proposed in the literature. Cyst evacuation was performed with relief of symptoms and improved visual outcomes at follow-up. Conclusion: While cystic adenomas can require total resection for cure, RCCs can show marked improvement with partial resection and evacuation of its contents. An accurate preoperative diagnosis can lead the surgeon to opt for the best surgical approach.

A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells.

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

Supraorbital transciliary keyhole approach for removal of tuberculum sellae meningioma: 3D surgical video.

BACKGROUND: Tuberculum sellae meningiomas have an incidence from 5 to 10% of all intracranial meningiomas[2] and tend to be surgically difficult and challenging tumors given their proximity to important structures such as the internal carotid artery (ICA), anterior cerebral artery (ACA), and optic nerves.[3] Typically, their growth is posteriorly and superiorly oriented, thereby displacing the optic nerves and causing visual dysfunction, which is the primary indication for surgical treatment.[1] The main goals of the treatment are the preservation or restoration of visual abilities and a complete tumor resection.[1] Conventionally, surgical approaches to tuberculum meningiomas involve largely invasive extended bifrontal, interhemispheric, orbitozygomatic, pterional, and subfrontal eyebrow approaches. The supraorbital craniotomy, however, is a minimally invasive transcranial approach that offers a similar surgical corridor to conventional transcranial approaches, using a limited craniotomy and minimal brain retraction that can be used for tumoral and vascular pathologies,[4,5] offering added cosmetic outcomes.[1] We present the case of a patient undergoing a supraorbital transciliary craniotomy with a tuberculum sellae meningioma causing bitemporal hemianopsia. CASE DESCRIPTION: A 70-year-old female with chronic headaches and progressive vision loss and visual field deficit for about 1 year. On ophthalmological evaluation, she was able to fixate and follow objects with each eye, light perception was only present in the right eye, and the vision in the left eye was 0.2 decimal units. Her visual fields demonstrated severe campimetric deficits. Her extraocular movements were intact and bilateral pupils were equal, round, and reactive to light. MRI of the brain demonstrated tuberculum sellae meningioma with bilateral optic canal invasion, displacing the chiasm, and extending ≥180° around the medial ICA wall and anterior ACA wall. The patient underwent supraorbital transciliary keyhole approach for total resection of the tumor. Postoperatively, visual acuity and visual field were significantly improved. CONCLUSION: Performing a supraorbital transciliary keyhole craniotomy for tuberculum sellae meningiomas requires an adequate and meticulous preoperative planning to determine the optimal surgical corridor to the lesion. The use of supraorbital craniotomy is safe with good cosmetic results and potentially lower morbidity allowing for adequate exposure, resection, and release of neurovascular structures.

Cryptococcal meningitis presenting as anterior spinal cord syndrome with accessory nerve palsy in immunocompetent patient: A case report.

BACKGROUND: Cryptococcus has a tropism for the nervous system with a higher prevalence of infection in immunosuppressed patients; it remains a major cause of human immunodeficiency virus (HIV)-related mortality worldwide. Neurological compromise caused by this microorganism mainly debuts as a meningeal syndrome, spinal involvement has been reported in literature, neuropathological assessments have found Cryptococci in spinal roots and meninges, with perineuritic adhesions probably explaining compromise lower cranial nerves and even spinal nerve roots. CASE DESCRIPTION: 39-year-old male seronegative for HIV, with a surgical history of hydrocephalus treated with ventriculoperitoneal shut 1 year before, he presented with progressive weakness in the four extremities evolving to be disabling with bilateral accessory nerve palsy and loss of sensation below his neck. The MR imaging showed diffuse leptomeningeal thickening both supra and infratentorial and over the spinal canal up to C5 with a cystic formation shown in the craniocervical union causing compression of the medullary bulb. The patient underwent a medial suboccipital craniectomy with resection of the posterior arch of c1 for sampling and decompression, pathologically appears numerous spherical organisms that have a thick clear capsule and are surrounded by histiocytes forming a granuloma compatible with Cryptococcus. Postoperatively, the patient's prior neurological deficits resolved. CONCLUSION: It is an infrequently suspected pathology in immunocompetent patients, usually requiring only antifungal treatment with adjustment of immunosuppressive or antiretroviral management. In special and rare situations like our case as presenting with lower cranial nerve and spinal involvement, surgical treatment is a priority for the resolution of the pathology and improves disabling neurological deficit.

A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2.

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

The use of exoscope combined with tubular retractor system for minimally invasive transsulcal resection of an ventricular atrium atypical choroid plexus papilloma: Three-dimensional operative video.

BACKGROUND: Choroid plexus papilloma represents 1-4% of pediatric brain tumors, mostly located in the ventricular atrium.[1] Intraventricular tumors represent a challenge due to the poor visualization of the surgical field and damage to surrounding structures.[2] Use of tubular retraction reduces cerebrovascular trauma to the surrounding parenchyma by distributing pressure uniformly, allowing less invasive corticotomy, and more stability on surgical corridors that allow the surgeon to use both hands and external visualization devices.[2-5]. CASE DESCRIPTION: We present the case of a 3-year-old boy with progressive headache, vomiting, and loss of control in the left hand for 3 months, with a history of ventricular shunt placement for acute obstructive hydrocephalus. The MRI revealed large lobulated lesion, which was hypointense on T1, hyperintense on T2, marked enhancement on T1 C+ (Gd) within the atrium of the right lateral ventricle, and spectroscopy with a peak of choline. Written consent for the use of photos and videos on this work was obtained from the patient's mother. A high-definition two-dimensional exoscope (VITOM® Karl Storz, Tuttlingen) was used during the surgical approach and throughout tumor removal, which was aided by ViewSite Brain Access System (VBAS®; Vycor Medical Inc.).[3] We performed a transparietal minimally invasive transsulcal parafascicular approach through the Frazier point for direct access to the ventricular atrium. Histological examination confirmed atypical choroid plexus papilloma. Postoperative imaging shows no residual tumor. The postoperative course was satisfactory with improvement of the headache and control of the left hand, leading to discharge home 1 week after surgery. CONCLUSION: The tubular transparietal minimally invasive approach obviates the need for traditional approaches to the atrium. This technique is safe and effective for the treatment of intraventricular and periventricular lesions, thus making this challenging target in more accessible to neurosurgeons, avoiding structure damage and any associated morbidity or mortality.

Robust production of uniform human cerebral organoids from pluripotent stem cells.

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human-specific cortical development and pathophysiology in an experimentally tractable system. Although diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we have directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single-cell profiling that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

Intrasellar cysticercosis cyst treated with a transciliary supraorbital keyhole approach - A case report.

BACKGROUND: Neurocysticercosis is the most common parasitic disease affecting the central nervous system. Isolated sellar cysticercosis cysts are rare and can mimic other sellar lesion as cystic pituitary adenoma, arachnoid cyst, Rathke cleft cyst, or craniopharyngioma. The surgical resection is mandatory because the cysticidal drugs are ineffective, however, new microsurgical approaches are emerging to reduce complications and need to test in this condition. We present a patient with a sellar cysticercosis cyst treated by transciliar supraorbital keyhole approach. CASE DESCRIPTION: A 45-year-old female with presented with chronic severe headaches, progressive deterioration of 6 months in visual acuity and bitemporal hemianopia. The pituitary hormonal levels were normal. Magnetic resonance findings showed a sellar and suprasellar cyst and underwent a microsurgical supraorbital transciliar keyhole approach for lesion resection. Pathologically, the lesion demonstrated a parasitic wall characterized by wavy, dense cuticle, and focal globular structure, surrounding inflammatory reaction with plasma cells. Postoperatively, the patient recovery fully neurologically. CONCLUSION: Intrasellar cysticercosis cyst causes significant neurological deficits due to its proximity to the chiasm, optic nerves, pituitary stalk, and the pituitary gland. Surgical section is an effective treatment. The supraorbital keyhole craniotomy offers satisfactory exposure, possibility of total resection with dissection of the supra and parasellar structures, short operative time, less blood loss, short hospital stay, and good overall surgical outcome.

Giving Drugs a Second Chance: Overcoming Regulatory and Financial Hurdles in Repurposing Approved Drugs As Cancer Therapeutics.

The repositioning or "repurposing" of existing therapies for alternative disease indications is an attractive approach that can save significant investments of time and money during drug development. For cancer indications, the primary goal of repurposed therapies is on efficacy, with less restriction on safety due to the immediate need to treat this patient population. This report provides a high-level overview of how drug developers pursuing repurposed assets have previously navigated funding efforts, regulatory affairs, and intellectual property laws to commercialize these "new" medicines in oncology. This article provides insight into funding programs (e.g., government grants and philanthropic organizations) that academic and corporate initiatives can leverage to repurpose drugs for cancer. In addition, we highlight previous examples where secondary uses of existing, Food and Drug Administration- or European Medicines Agency-approved therapies have been predicted in silico and successfully validated in vitro and/or in vivo (i.e., animal models and human clinical trials) for certain oncology indications. Finally, we describe the strategies that the pharmaceutical industry has previously employed to navigate regulatory considerations and successfully commercialize their drug products. These factors must be carefully considered when repurposing existing drugs for cancer to best benefit patients and drug developers alike.