Increased demand for NAD+ relative to ATP drives aerobic glycolysis.

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD+/NADH ratio. This change in NAD+/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD+ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD+ is in excess of the demand for ATP.

Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

Low HBV knowledge is associated with low HBV vaccination uptake in general adult population despite incentivization of HBV vaccination.

BACKGROUND: Hepatitis B virus (HBV) vaccination in Vietnamese adults remains low and unequally distributed. We conducted a study on HBV-naïve adults living in Ho Chi Minh City, Viet Nam, to determine barriers associated with HBV vaccination uptake after removing the financial barrier by providing free coupons for HBV vaccination. METHODS: After being screened for HBsAg, anti-HBs, and anti-HBc, 284 HBV-naïve study participants aged 18 and over (i.e., negative for HBsAg, anti-HBs, and anti-HBc total) were provided free 3-dose HBV vaccine coupons. Next, study participants' receipt of 1st, 2nd, and 3rd doses of HBV vaccine was documented at a pre-specified study healthcare facility, where HBV vaccines were distributed at no cost to the participants. Upon study entry, participants answered questionnaires on sociodemographics, knowledge of HBV and HBV vaccination, and related social and behavioral factors. The proportions of three doses of HBV vaccine uptake and their confidence intervals were analyzed. Associations of HBV vaccine initiation with exposures at study entry were evaluated using modified Poisson regression. RESULTS: 98.9% (281 of 284) of study participants had complete data and were included in the analysis. The proportion of participants obtaining the 1st, 2nd, and 3rd doses of HBV vaccine was 11.7% (95% Confidence Interval [95% CI] 8.0-15.5%), 10.7% (95%CI 7.1-14.3%), and 8.9% (95%CI 5.6-12.2%), respectively. On the other hand, participants were more likely to initiate the 1st dose if they had adequate knowledge of transmission (adjusted relative risk [aRR] = 2.58, 95% CI 1.12-5.92), adequate knowledge of severity (aRR = 6.75, 95%CI 3.38-13.48), and annual health-checking seeking behavior (aRR = 2.04, 95%CI 1.07-3.87). CONCLUSION: We documented a low HBV vaccination uptake despite incentivization. However, increased vaccine initiation was associated with better HBV knowledge and annual health check-up adherence. When considering expanding HBV vaccination to the general adult population, we should appreciate that HBV knowledge is an independent predictor of vaccine uptake.

Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma.

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

Effects of the KCNQ channel opener ezogabine on functional connectivity of the ventral striatum and clinical symptoms in patients with major depressive disorder.

Major depressive disorder (MDD) is a leading cause of disability worldwide, yet current treatment strategies remain limited in their mechanistic diversity. Recent evidence has highlighted a promising novel pharmaceutical target-the KCNQ-type potassium channel-for the treatment of depressive disorders, which may exert a therapeutic effect via functional changes within the brain reward system, including the ventral striatum. The current study assessed the effects of the KCNQ channel opener ezogabine (also known as retigabine) on reward circuitry and clinical symptoms in patients with MDD. Eighteen medication-free individuals with MDD currently in a major depressive episode were enrolled in an open-label study and received ezogabine up to 900 mg/day orally over the course of 10 weeks. Resting-state functional magnetic resonance imaging data were collected at baseline and posttreatment to examine brain reward circuitry. Reward learning was measured using a computerized probabilistic reward task. After treatment with ezogabine, subjects exhibited a significant reduction of depressive symptoms (Montgomery-Asberg Depression Rating Scale score change: -13.7 ± 9.7, p < 0.001, d = 2.08) and anhedonic symptoms (Snaith-Hamilton Pleasure Scale score change: -6.1 ± 5.3, p < 0.001, d = 1.00), which remained significant even after controlling for overall depression severity. Improvement in depression was associated with decreased functional connectivity between the ventral caudate and clusters within the mid-cingulate cortex and posterior cingulate cortex (n = 14, voxel-wise p < 0.005). In addition, a subgroup of patients tested with a probabilistic reward task (n = 9) showed increased reward learning following treatment. These findings highlight the KCNQ-type potassium channel as a promising target for future drug discovery efforts in mood disorders.

irCLIP platform for efficient characterization of protein-RNA interactions.

The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.

Dissecting noncoding and pathogen RNA-protein interactomes.

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

Sickle cell disease and the eye.

PURPOSE OF REVIEW: To review recent literature pertaining to sickle cell retinopathy (SCR) and, in particular, sickle cell maculopathy. RECENT FINDINGS: Several recent studies suggest that macular perfusion abnormalities seen in patients with sickle cell disease of various genotypes may affect both the superficial and deep capillary plexi, with a predilection for the deep capillary plexus. Further, these changes may be associated with areas of macular thinning, as well as with peripheral retinal ischemia, even in individuals without visual symptoms, contrary to what has previously been described in both diabetic retinopathy and retinal vein occlusion. Several cases also suggest that paracentral acute middle maculopathy may be the pathophysiologic mechanism by which microvascular occlusion leads to macular thinning. SUMMARY: Sickle cell disease can manifest in a number of ways within the orbit as well as intraocularly because of its nonspecific vasoocclusive episodes. However, SCR is the most common ophthalmic manifestation of this disease. Historically, SCR has been considered a peripheral retinopathy, but the development and use of spectral-domain optical coherence tomography and optical coherence tomography angiography suggest that significant macular vascular changes occur early in this disease, even in asymptomatic individuals.

Stiffness Change for Reconfiguration of Inflated Beam Robots.

Abstract Active control of the shape of soft robots is challenging. Despite having an infinite number of passive degrees of freedom (DOFs), soft robots typically only have a few actively controllable DOFs, limited by the number of degrees of actuation (DOAs). The complexity of actuators restricts the number of DOAs that can be incorporated into soft robots. Active shape control is further complicated by the buckling of soft robots under compressive forces; this is particularly challenging for compliant continuum robots due to their long aspect ratios. In this study, we show how variable stiffness enables shape control of soft robots by addressing these challenges. Dynamically changing the stiffness of sections along a compliant continuum robot selectively "activates" discrete joints. By changing which joints are activated, the output of a single actuator can be reconfigured to actively control many different joints, thus decoupling the number of controllable DOFs from the number of DOAs. We demonstrate embedded positive pressure layer jamming as a simple method for stiffness change in inflated beam robots, its compatibility with growing robots, and its use as an "activating" technology. We experimentally characterize the stiffness change in a growing inflated beam robot and present finite element models that serve as guides for robot design and fabrication. We fabricate a multisegment everting inflated beam robot and demonstrate how stiffness change is compatible with growth through tip eversion, enables an increase in workspace, and achieves new actuation patterns not possible without stiffening.

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress.

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

Placement of ProKera in the management of ocular manifestations of acute Stevens-Johnson syndrome in an outpatient.

PURPOSE: To report the clinical use of ProKera (Bio-Tissue, Inc., Miami, FL) under topical anesthesia in an outpatient for the management of ocular manifestations of acute Stevens-Johnson syndrome (SJS). METHODS: Interventional case report. RESULTS: A 19-year-old woman developed acute SJS with ocular involvement after ingestion of oral antibiotics. Slit-lamp examination performed 2 weeks later showed severe inflammation and areas of ulceration along all 4 lids and complete, bilateral sloughing of bulbar and palpebral conjunctivae, including the limbus. ProKera was placed under topical anesthesia without sedation in both eyes instead of an amniotic membrane due to excess risk of general anesthesia. Three weeks after placement, slit-lamp examination showed complete re-epithelialization of both corneas and conjunctivae, with only trace conjunctival injection and minor limbal epithelial irregularities. Three months postprocedure, there were no signs of clinically significant scarring, and the visual acuity (VA) was 20/20 bilaterally. Fourteen months postprocedure, VA remained stable, and the patient did not have dry eye, photophobia, clinically significant scarring, or symblepharon. CONCLUSIONS: ProKera placement performed under topical anesthesia may be appropriate for the treatment of ocular surface manifestations of acute SJS particularly in those patients followed in an outpatient setting with milder forms of disease and/or with contraindications to general anesthesia.

Posterior placoid-like maculopathy and macular hole associated with vitamin A deficiency.

Purpose: To report a case of bilateral posterior placoid-like maculopathy and a macular hole associated with vitamin A deficiency. Observations: A 72-year-old male presented with nyctalopia and progressive vision loss in both eyes. Examination and multimodal imaging were consistent with posterior placoid-like maculopathy bilaterally and a macular hole in the right eye. A workup for infectious, inflammatory, and paraneoplastic etiologies revealed a severely low serum vitamin A level. Two months after initiation of vitamin A repletion, there was improvement in best-corrected Snellen visual acuity as well as macular hole closure. A diagnosis of posterior placoid-like maculopathy in the setting of vitamin A deficiency (VAD) was made. Conclusions and importance: VAD should be considered when symmetric posterior pole placoid-like lesions are observed and other, more common etiologies have been ruled out.

Zanubrutinib in Mantle Cell Lymphoma Management: A Comprehensive Review.

Purpose: The pharmacology, pharmacokinetics, pharmacodynamics, clinical efficacy, and safety of zanbrutinib are described. Summary: Mantle cell lymphoma (MCL) is a mature B-cell lymphoma that is typically associated with unfavorable outcomes, and virtually all patients with MCL have refractory or relapsed disease despite aggressive treatment. The treatment paradigm for MCL has transformed dramatically over the past decade owing to rapid advancements in immunotherapy and molecular-targeted therapies. Zanubrutinib, a second-generation Bruton's tyrosine kinase inhibitor (BTKI) designated for mature B-cell non-Hodgkin's lymphoma (NHL), has drastically improved the survival outcomes in relapsed/refractory (R/R) MCL patients. This selective BTKI is a small molecule that functions by forming a covalent bond in the active site of BTK. The inhibition of BTK activity is essential for the signaling of B-cell antigen receptor (BCR) and cytokine receptor pathways. In a preclinical study, zanubrutinib inhibited malignant B-cell proliferation and reduced tumor growth. Zanubrutinib was granted FDA-accelerated approval based on the results of Phase I and II trials. The investigator-assessed overall response rate was 83.7%, of which 78% of patients achieved complete response. The median duration of response was 19.5 months, and the median progression-free survival was 22.1 months. The most common (≥20%) all-grade adverse events were low neutrophil count (46.5%), upper respiratory tract infection (38.4%), rash (36.0%), low white blood cell count (33.7%), and low platelet count (32.6%). Conclusion: Zanubrutinib is a selective, next-generation, orally active, irreversible BTK inhibitor. The selectivity of zanubrutinib and its superior efficacy, with a well-tolerated safety profile, have proven to be attractive options for other malignancies.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

A case of ustekinumab-induced sclerouveitis.

PURPOSE: Biologics have proven to be essential therapeutic agents in immune-mediated systemic diseases. Ophthalmologic adverse effects have been reported in the use of more traditional agents, such as TNF-α inhibitors, but there are limited data on ocular side effects associated with the newer generation of biologics. OBSERVATIONS: In this report, we present a case of a suspected ustekinumab-associated recurrent sclerouveitis. CONCLUSIONS: To the best of our knowledge, this is the first reported case of this type seen with ustekinumab injections. Our goal is to add to the existing literature in order to better understand the mechanism and management of this condition.

GOT2 consider the tumor microenvironment.

To thrive in a hypoxic and nutrient-limited tumor microenvironment, pancreatic ductal adenocarcinoma (PDAC) cells rewire their metabolism. Understanding PDAC cell metabolism may uncover vulnerabilities that can be targeted for improved therapy. Three recent studies find that the PDAC tumor microenvironment modulates the functional consequences of depleting the mitochondrially localized aspartate transaminase GOT2, thus providing new insights into the metabolism of this lethal cancer.

Association of Dupilumab with Intraocular Inflammation.

PURPOSE: To report four cases of uveitis after treatment with dupilumab for atopic dermatitis. METHODS: Retrospective case series. RESULTS: Data was collected from four patients with inflammation that developed after treatment with dupilumab. The first patient was thought to have developed posterior scleritis or Harada's-type disease related to her treatment with dupilumab, with recurrence of intraocular inflammation upon restarting treatment. The second patient developed anterior and intermediate uveitis as well as cystoid macular edema in her right eye about two years after starting dupilumab treatment. The third patient developed a distinct relentless placoid chorioretinitis while on therapy with dupilumab. The fourth patient developed bilateral cystoid macular edema while on treatment with dupilumab. CONCLUSION: We report a case series of patients treated with dupilumab who developed intraocular manifestations of inflammatory disease.

Describing Adverse Events Associated with Bilateral Same-Day Intravitreal Dexamethasone Implants.

BACKGROUND AND OBJECTIVES: To explore the incidence of adverse events after bilateral same-day intravitreal 0.7-mg dexamethasone implant (SDIDI) injections. MATERIALS AND METHODS: We performed an IRB approved, single-center, retrospective review of patients receiving bilateral SDIDI injections from January 1, 2016 to October 31, 2021 and reviewed adverse events that occurred within 3 months of injection. RESULTS: A total of 206 bilateral (412 eyes) SDIDI injections were performed in 59 patients. Ocular hypertension or the addition of intraocular pressure (IOP) lowering drops occurred in 121 (29.4%) eyes after IDI. Two (0.5%) eyes required glaucoma drainage surgeries. Of the 117 phakic eyes, 32 (27.4%) had progression of cataract or cataract extraction. There were two (0.5%) episodes of vitreous hemorrhage and one (0.2%) retinal tear with retinal detachment. There were no cases of endophthalmitis. CONCLUSION: Serious complication rates after bilateral same-day IDI injections appears low. Increased IOP that requires intervention can occur. [Ophthalmic Surg Lasers Imaging Retina 2022;53:612-618.].

Nucleotide imbalance decouples cell growth from cell proliferation.

Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.