Identifying the Mechanisms of a Peripherally Limited Exercise Phenotype in Patients With Heart Failure With Preserved Ejection Fraction.

BACKGROUND: We identified peripherally limited patients using cardiopulmonary exercise testing and measured skeletal muscle oxygen transport and utilization during invasive single leg exercise testing to identify the mechanisms of the peripheral limitation. METHODS: Forty-five patients with heart failure with preserved ejection fraction (70±7 years, 27 females) completed seated upright cardiopulmonary exercise testing and were defined as having a (1) peripheral limitation to exercise if cardiac output/oxygen consumption (VO2) was elevated (≥6) or 5 to 6 with a stroke volume reserve >50% (n=31) or (2) a central limitation to exercise if cardiac output/VO2 slope was ≤5 or 5 to 6 with stroke volume reserve <50% (n=14). Single leg knee extension exercise was used to quantify peak leg blood flow (Doppler ultrasound), arterial-to-venous oxygen content difference (femoral venous catheter), leg VO2, and muscle oxygen diffusive conductance. In a subset of participants (n=36), phosphocreatine recovery time was measured by magnetic resonance spectroscopy to determine skeletal muscle oxidative capacity. RESULTS: Peak VO2 during cardiopulmonary exercise testing was not different between groups (central: 13.9±5.7 versus peripheral: 12.0±3.1 mL/min per kg; P=0.135); however, the peripheral group had a lower peak arterial-to-venous oxygen content difference (central: 13.5±2.0 versus peripheral: 11.1±1.6 mLO2/dL blood; P<0.001). During single leg knee extension, there was no difference in peak leg VO2 (P=0.306), but the peripherally limited group had greater blood flow/VO2 ratio (P=0.024), lower arterial-to-venous oxygen content difference (central: 12.3±2.5 versus peripheral: 10.3±2.2 mLO2/dL blood; P=0.013), and lower muscle oxygen diffusive conductance (P=0.021). A difference in magnetic resonance spectroscopy-derived phosphocreatine recovery time was not detected (P=0.199). CONCLUSIONS: Peripherally limited patients with heart failure with preserved ejection fraction identified by cardiopulmonary exercise testing have impairments in oxygen transport and utilization at the level of the skeletal muscle quantified by invasive knee extension exercise testing, which includes an increased blood flow/V̇O2 ratio and poor muscle diffusive capacity. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04068844.

DCS blood flow index underestimates skeletal muscle perfusion in vivo: rationale and early evidence for the NIRS-DCS perfusion index.

Significance: Diffuse correlation spectroscopy (DCS) permits non-invasive assessment of skeletal muscle blood flow but may misestimate changes in muscle perfusion. Aim: We aimed to highlight recent evidence that DCS blood flow index (BFI) misestimates changes in muscle blood flow during physiological perturbation and to introduce a novel approach that adjusts BFI for estimated changes in vasodilation. Approach: We measured changes in muscle BFI during quadriceps and forearm exercises using DCS, the latter of which were adjusted for estimated changes in microvascular flow area and then compared to Doppler ultrasound in the brachial artery. Then, we compared adjusted BFI- and arterial spin labeling (ASL) MRI measures of gastrocnemius blood flow during reactive hyperemia and plantar flexion exercise. Results: We observed little-to-no change in quadriceps BFI during maximal-effort exercise. Similarly, forearm BFI was modestly increased during handgrip exercise, but the magnitude was significantly lower than measured by Doppler ultrasound in the brachial artery. However, this difference was ameliorated after adjusting BFI for estimated changes in microvascular flow area. Similar observations were also observed in the gastrocnemius when directly comparing the adjusted BFI values to ASL-MRI. Conclusions: Adjusting BFI for estimated changes in microvascular flow area may improve DCS estimates of muscle blood flow, but further study is needed to validate these methods moving forward.

Measurements of in vivo skeletal muscle oxidative capacity are lower following sustained isometric compared with dynamic contractions.

Human skeletal muscle oxidative capacity can be quantified non-invasively using 31-phosphorus magnetic resonance spectroscopy (31P-MRS) to measure the rate constant of phosphocreatine (PCr) recovery (kPCr) following contractions. In the quadricep muscles, several studies have quantified kPCr following 24-30 s of sustained maximal voluntary isometric contraction (MVIC). This approach has the advantage of simplicity but is potentially problematic because sustained MVICs inhibit perfusion, which may limit muscle oxygen availability or increase the intracellular metabolic perturbation, and thus affect kPCr. Alternatively, dynamic contractions allow reperfusion between contractions, which may avoid limitations in oxygen delivery. To determine whether dynamic contraction protocols elicit greater kPCr than sustained MVIC protocols, we used a cross-sectional design to compare quadriceps kPCr in 22 young and 11 older healthy adults following 24 s of maximal voluntary: (1) sustained MVIC and (2) dynamic (MVDC; 120°·s-1, 1 every 2 s) contractions. Muscle kPCr was ∼20% lower following the MVIC protocol compared with the MVDC protocol (p ≤ 0.001), though this was less evident in older adults (p = 0.073). Changes in skeletal muscle pH (p ≤ 0.001) and PME accumulation (p ≤ 0.001) were greater following the sustained MVIC protocol, and pH (p ≤ 0.001) and PME (p ≤ 0.001) recovery were slower. These results demonstrate that (i) a brief, sustained MVIC yields a lower value for skeletal muscle oxidative capacity than an MVDC protocol of similar duration and (ii) this difference may not be consistent across populations (e.g., young vs. old). Thus, the potential effect of contraction protocol on comparisons of kPCr in different study groups requires careful consideration in the future.

Automatic Classification of Slit-Lamp Photographs by Imaging Illumination.

PURPOSE: The aim of this study was to facilitate deep learning systems in image annotations for diagnosing keratitis type by developing an automated algorithm to classify slit-lamp photographs (SLPs) based on illumination technique. METHODS: SLPs were collected from patients with corneal ulcer at Kellogg Eye Center, Bascom Palmer Eye Institute, and Aravind Eye Care Systems. Illumination techniques were slit beam, diffuse white light, diffuse blue light with fluorescein, and sclerotic scatter (ScS). Images were manually labeled for illumination and randomly split into training, validation, and testing data sets (70%:15%:15%). Classification algorithms including MobileNetV2, ResNet50, LeNet, AlexNet, multilayer perceptron, and k-nearest neighborhood were trained to distinguish 4 type of illumination techniques. The algorithm performances on the test data set were evaluated with 95% confidence intervals (CIs) for accuracy, F1 score, and area under the receiver operator characteristics curve (AUC-ROC), overall and by class (one-vs-rest). RESULTS: A total of 12,132 images from 409 patients were analyzed, including 41.8% (n = 5069) slit-beam photographs, 21.2% (2571) diffuse white light, 19.5% (2364) diffuse blue light, and 17.5% (2128) ScS. MobileNetV2 achieved the highest overall F1 score of 97.95% (CI, 97.94%-97.97%), AUC-ROC of 99.83% (99.72%-99.9%), and accuracy of 98.98% (98.97%-98.98%). The F1 scores for slit beam, diffuse white light, diffuse blue light, and ScS were 97.82% (97.80%-97.84%), 96.62% (96.58%-96.66%), 99.88% (99.87%-99.89%), and 97.59% (97.55%-97.62%), respectively. Slit beam and ScS were the 2 most frequently misclassified illumination. CONCLUSIONS: MobileNetV2 accurately labeled illumination of SLPs using a large data set of corneal images. Effective, automatic classification of SLPs is key to integrating deep learning systems for clinical decision support into practice workflows.

The long head of the biceps tendon undergoes multiaxial deformation during shoulder motion.

The long head biceps tendon (LHBT) is presumed a common source of shoulder joint pain and injury. Despite common LHBT pathologies, diagnosis and preferred treatment remain frequently debated. This Short Communication reports the development of a subject-specific finite element model of the shoulder joint based on one subject's 3D reconstructed anatomy and 3D in vivo kinematics recorded from bone-fixed electromagnetic sensors. The primary purpose of this study was to use the developed finite element model to investigate the LHBT mechanical environment during a typical shoulder motion of arm raising. Furthermore, this study aimed to assess the viability of material models derived from uniaxial tensile tests for accurate simulation of in vivo motion. The findings of our simulations indicate that the LHBT undergoes complex multidimensional deformations. As such, uniaxial material properties reported in the existing body of literature are not sufficient to simulate accurately the in vivo mechanical behavior of the LHBT. Further experimental tests on cadaveric specimens, such as biaxial tension and combinations of tension and torsion, are needed to describe fully the mechanical behavior of the LHBT and investigate its mechanisms of injury.

Muscle fatigue, bioenergetic responses and metabolic economy during load- and velocity-based maximal dynamic contractions in young and older adults.

We evaluated whether task-dependent, age-related differences in muscle fatigue (contraction-induced decline in normalized power) develop from differences in bioenergetics or metabolic economy (ME; mass-normalized work/mM ATP). We used magnetic resonance spectroscopy to quantify intracellular metabolites in vastus lateralis muscle of 10 young and 10 older adults during two maximal-effort, 4-min isotonic (20% maximal torque) and isokinetic (120°s-1 ) contraction protocols. Fatigue, inorganic phosphate (Pi), and pH (p ≥ 0.213) differed by age during isotonic contractions. However, older had less fatigue (p ≤ 0.011) and metabolic perturbation (lower [Pi], greater pH; p ≤ 0.031) than young during isokinetic contractions. ME was lower in older than young during isotonic contractions (p ≤ 0.003), but not associated with fatigue in either protocol or group. Rather, fatigue during both tasks was linearly related to changes in [H+ ], in both groups. The slope of fatigue versus [H+ ] was 50% lower in older than young during isokinetic contractions (p ≤ 0.023), consistent with less fatigue in older during this protocol. Overall, regardless of age or task type, acidosis, but not ME, was the primary mechanism for fatigue in vivo. The source of the age-related differences in contraction-induced acidosis in vivo remains to be determined, as does the apparent task-dependent difference in the sensitivity of muscle to [H+ ].

Developmental regulators moonlighting as transposons defense factors.

BACKGROUND: The germline perpetuates genetic information across generations. To maintain the integrity of the germline, transposable elements in the genome must be silenced, as these mobile elements would otherwise engender widespread mutations passed on to subsequent generations. There are several well-established mechanisms that are dedicated to providing defense against transposable elements, including DNA methylation, RNA interference, and the PIWI-interacting RNA pathway. OBJECTIVES: Recently, several studies have provided evidence that transposon defense is not only provided by factors dedicated to this purpose but also factors with other roles, including in germline development. Many of these are transcription factors. Our objective is to summarize what is known about these "bi-functional" transcriptional regulators. MATERIALS AND METHODS: Literature search. RESULTS AND CONCLUSION: We summarize the evidence that six transcriptional regulators-GLIS3, MYBL1, RB1, RHOX10, SETDB1, and ZBTB16-are both developmental regulators and transposable element-defense factors. These factors act at different stages of germ cell development, including in pro-spermatogonia, spermatogonial stem cells, and spermatocytes. Collectively, the data suggest a model in which specific key transcriptional regulators have acquired multiple functions over evolutionary time to influence developmental decisions and safeguard transgenerational genetic information. It remains to be determined whether their developmental roles were primordial and their transposon defense roles were co-opted, or vice versa.

Targeting nonsense-mediated RNA decay does not increase progranulin levels in the Grn R493X mouse model of frontotemporal dementia.

A common cause of frontotemporal dementia (FTD) are nonsense mutations in the progranulin (GRN) gene. Because nonsense mutations activate the nonsense-mediated RNA decay (NMD) pathway, we sought to inhibit this RNA turnover pathway as a means to increase progranulin levels. Using a knock-in mouse model harboring a common patient mutation, we tested whether either pharmacological or genetic inhibition of NMD upregulates progranulin in these GrnR493X mice. We first examined antisense oligonucleotides (ASOs) targeting an exonic region in GrnR493X mRNA predicted to block its degradation by NMD. As we previously reported, these ASOs effectively increased GrnR493X mRNA levels in fibroblasts in vitro. However, following CNS delivery, we found that none of the 8 ASOs we tested increased Grn mRNA levels in the brains of GrnR493X mice. This result was obtained despite broad ASO distribution in the brain. An ASO targeting a different mRNA was effective when administered in parallel to wild-type mice. As an independent approach to inhibit NMD, we examined the effect of loss of an NMD factor not required for embryonic viability: UPF3b. We found that while Upf3b deletion effectively perturbed NMD, it did not increase Grn mRNA levels in Grn+/R493X mouse brains. Together, our results suggest that the NMD-inhibition approaches that we used are likely not viable for increasing progranulin levels in individuals with FTD caused by nonsense GRN mutations. Thus, alternative approaches should be pursued.

Epinephrine iontophoresis attenuates changes in skin blood flow and abolishes cutaneous contamination of near-infrared diffuse correlation spectroscopy estimations of muscle perfusion.

Near-infrared diffuse correlation spectroscopy (NIR-DCS) is an optical imaging technique for measuring relative changes in skeletal muscle microvascular perfusion (i.e., fold change above baseline) during reactive hyperemia testing and exercise and is reported as a blood flow index (BFI). Although it is generally accepted that changes in BFI are primarily driven by changes in muscle perfusion, it is well known that large, hyperthermia-induced changes in cutaneous blood flow can uncouple this relationship. What remains unknown, is how much of an impact that changes in cutaneous perfusion have on NIR-DCS BFI and estimates of skeletal muscle perfusion under thermoneutral conditions, where changes in cutaneous blood flow are assumed to be relatively low. We therefore used epinephrine iontophoresis to pharmacologically block changes in cutaneous perfusion throughout a battery of experimental procedures. The data show that 1) epinephrine iontophoresis attenuates changes in cutaneous perfusion for up to 4-h posttreatment, even in the face of significant neural and local stimuli, 2) under thermoneutral conditions, cutaneous perfusion does not significantly impact NIR-DCS BFI during reactive hyperemia testing or moderate-intensity exercise, and 3) during passive whole body heat stress, when cutaneous vasodilation is pronounced, epinephrine iontophoresis preserves NIR-DCS measures of skeletal muscle BFI during moderate-intensity exercise. Collectively, these data suggest that cutaneous perfusion is unlikely to have a major impact on NIR-DCS estimates of skeletal muscle BFI under thermoneutral conditions, but that epinephrine iontophoresis can be used to abolish cutaneous contamination of the NIR-DCS BFI signal during studies where skin blood flow may be elevated but skeletal muscle perfusion is of specific interest.

Microbial Keratitis Isolates at a Midwestern Tertiary Eye Care Center.

PURPOSE: The aim of the study was to describe the pathogen, antimicrobial susceptibility, and trends over time of microbial keratitis (MK) at a Midwestern tertiary eye center. METHODS: Patients with MK were identified in the electronic health record from August 2012 to December 2021. Diagnostic laboratory tests with an MK diagnosis were identified and classified as laboratory positive or laboratory negative. Laboratory-positive infections were categorized as bacterial (gram-positive, gram-negative, or acid-fast bacilli), fungal, viral, Acanthamoeba , or polymicrobial. Antimicrobial susceptibilities were obtained. Trends over time were assessed using linear regression. RESULTS: Of 3288 patients with MK identified, 1012 (30.8%) had laboratory tests performed. Laboratory-positive infections (n = 499, 49.3%) were bacterial in 73.5% (n = 367) of cases, fungal in 7.8% (n = 39), viral in 1.6% (n = 8), Acanthamoeba in 1.4% (n = 7), and polymicrobial in 15.6% (n = 78). Of bacterial infections, 70% (n = 257) were gram-positive, with coagulase-negative Staphylococcus (CoNS; 31%) and Staphylococcus aureus ( S. aureus ; 23%) as the most common pathogens. Bacteria were acid-fast bacilli in 1.9% (n = 7) of cases and gram-negative in 28.1% (n = 103), with Pseudomonas aeruginosa as the predominant pathogen (47.7%). S. aureus showed antibiotic resistance from 0% (vancomycin and gentamicin) to 50% (erythromycin); CoNS from 0% (vancomycin, gentamicin, and moxifloxacin) to 64% (erythromycin). The rate of laboratory-negative MK significantly increased over time (slope estimate = 2.1% per year, P = 0.034). Rates of bacterial, fungal, viral, Acanthamoeba , and polymicrobial infections were stable over time (all slope P > 0.05). CONCLUSIONS: Bacterial keratitis accounted for most MK cases. Gram-positive bacteria were the most common isolates. CoNS and S. aureus were universally susceptible to vancomycin. Rates of MK infection types were stable over time.

Evaluation of different getter substrates using two-dimensional gas chromatography with time of flight mass spectrometry.

While understanding hydrogen uptake by organic based getters such as 1,4-bis(phenylethynyl)benzene (DEB) combined with a palladium(0)bis(dibenzylideneacetone) (Pd(dba)2) catalyst is essential, another crucial element to understand is the decomposition of the DEB, Pd(dba)2, and/or substrate material. The breakdown of these materials may create unwanted volatiles, which may interact with and lead to deterioration of sensitive materials. Moreover, it is critical to understand if different substrates cause the getter and/or catalyst to degrade in different manners. Utilizing comprehensive two-dimensional gas chromatography (GC×GC) with time-of-flight mass spectrometry (TOFMS), the presence of volatiles located in the headspace of various DEB/Pd(dba)2 getter substrates is examined. These samples include a getter infused silicone foam, a hydrogenated getter infused silicone foam, an activated carbon getter pellet, and a hydrogenated activated carbon getter pellet. Application of Fisher ratio (F-ratio) analyses lead to the identification of several compounds that are generated or consumed through the hydrogenation process. These include benzene derivatives such as bibenzyl, benzaldehyde, and vinyl benzoate in the activated carbon pellets and 1,5-diphenyl-3-pentanone, toluene, styrene, and 1-1'(2-pentene 1,5-diyl)bis benzene in the silicone foams, and alkane/alkene derivatives such undecane, 4-tridecene, and decane in the activated carbon pellets and 2,6-dimethyl undecane in the silicone foams. Further comparison of the different hydrogenated getter substrates (e.g. activated carbon pellet and silicone foam) indicates that the different substrates alter the decomposition products created from the degradation of the DEB and Pd(dba)2.

Progression of the pluripotent epiblast depends upon the NMD factor UPF2.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that degrades RNAs harboring in-frame stop codons in specific contexts. Loss of NMD factors leads to embryonic lethality in organisms spanning the phylogenetic scale, but the mechanism remains unknown. Here, we report that the core NMD factor, UPF2, is required for expansion of epiblast cells within the inner cell mass of mice in vivo. We identify NMD target mRNAs in mouse blastocysts - both canonical and alternatively processed mRNAs - including those encoding cell cycle arrest and apoptosis factors, raising the possibility that NMD is essential for embryonic cell proliferation and survival. In support, the inner cell mass of Upf2-null blastocysts rapidly regresses with outgrowth and is incompetent for embryonic stem cell derivation in vitro. In addition, we uncovered concordant temporal- and lineage-specific regulation of NMD factors and mRNA targets, indicative of a shift in NMD magnitude during peri-implantation development. Together, our results reveal developmental and molecular functions of the NMD pathway in the early embryo.

Quantifying Clinicians' Diagnostic Uncertainty When Making Initial Treatment Decisions for Microbial Keratitis.

PURPOSE: There is a need to understand physicians' diagnostic uncertainty in the initial management of microbial keratitis (MK). This study aimed to understand corneal specialists' diagnostic uncertainty by establishing risk thresholds for treatment of MK that could be used to inform a decision curve analysis for prediction modeling. METHODS: A cross-sectional survey of corneal specialists with at least 2 years clinical experience was conducted. Clinicians provided the percentage risk at which they would always or never treat MK types (bacterial, fungal, herpetic, and amoebic) based on initial ulcer sizes and locations (<2 mm2 central, <2 mm2 peripheral, and >8 mm2 central). RESULTS: Seventy-two of 99 ophthalmologists participated who were 50% female with an average of 14.7 (SD = 10.1) years of experience, 60% in academic practices, and 38% outside the United States. Clinicians reported they would "never" and "always" treat a <2 mm2 central MK infection if the median risk was 0% and 20% for bacterial (interquartile range, IQR = 0-5 and 5-50), 4.5% and 27.5% for herpetic (IQR = 0-10 and 10-50), 5% and 50% for fungal (IQR = 0-10 and 20-75), and 5% and 50.5% for amoebic (IQR = 0-20 and 32-80), respectively. Mixed-effects models showed lower thresholds to treat larger and central infections (P < 0.001, respectively), and thresholds to always treat differed between MK types for the United States (P < 0.001) but not international clinicians. CONCLUSIONS: Risk thresholds to treat differed by practice locations and MK types, location, and size. Researchers can use these thresholds to understand when a clinician is uncertain and to create decision support tools to guide clinicians' treatment decisions.

SMG6 localizes to the chromatoid body and shapes the male germ cell transcriptome to drive spermatogenesis.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA turnover pathway that depends on the endonuclease SMG6. Here, we show that SMG6 is essential for male germ cell differentiation in mice. Germ-cell conditional knockout (cKO) of Smg6 induces extensive transcriptome misregulation, including a failure to eliminate meiotically expressed transcripts in early haploid cells, and accumulation of NMD target mRNAs with long 3' untranslated regions (UTRs). Loss of SMG6 in the male germline results in complete arrest of spermatogenesis at the early haploid cell stage. We find that SMG6 is strikingly enriched in the chromatoid body (CB), a specialized cytoplasmic granule in male germ cells also harboring PIWI-interacting RNAs (piRNAs) and the piRNA-binding protein PIWIL1. This raises the possibility that SMG6 and the piRNA pathway function together, which is supported by several findings, including that Piwil1-KO mice phenocopy Smg6-cKO mice and that SMG6 and PIWIL1 co-regulate many genes in round spermatids. Together, our results demonstrate that SMG6 is an essential regulator of the male germline transcriptome, and highlight the CB as a molecular platform coordinating RNA regulatory pathways to control sperm production and fertility.

Regulation of both transcription and RNA turnover contribute to germline specification.

The nuanced mechanisms driving primordial germ cells (PGC) specification remain incompletely understood since genome-wide transcriptional regulation in developing PGCs has previously only been defined indirectly. Here, using SLAMseq analysis, we determined genome-wide transcription rates during the differentiation of embryonic stem cells (ESCs) to form epiblast-like (EpiLC) cells and ultimately PGC-like cells (PGCLCs). This revealed thousands of genes undergoing bursts of transcriptional induction and rapid shut-off not detectable by RNAseq analysis. Our SLAMseq datasets also allowed us to infer RNA turnover rates, which revealed thousands of mRNAs stabilized and destabilized during PGCLC specification. mRNAs tend to be unstable in ESCs and then are progressively stabilized as they differentiate. For some classes of genes, mRNA turnover regulation collaborates with transcriptional regulation, but these processes oppose each other in a surprisingly high frequency of genes. To test whether regulated mRNA turnover has a physiological role in PGC development, we examined three genes that we found were regulated by RNA turnover: Sox2, Klf2 and Ccne1. Circumvention of their regulated RNA turnover severely impaired the ESC-to-EpiLC and EpiLC-to-PGCLC transitions. Our study demonstrates the functional importance of regulated RNA stability in germline development and provides a roadmap of transcriptional and post-transcriptional regulation during germline specification.

SNPs, short tandem repeats, and structural variants are responsible for differential gene expression across C57BL/6 and C57BL/10 substrains.

Mouse substrains are an invaluable model for understanding disease. We compared C57BL/6J, which is the most commonly used inbred mouse strain, with eight C57BL/6 and five C57BL/10 closely related inbred substrains. Whole-genome sequencing and RNA-sequencing analysis yielded 352,631 SNPs, 109,096 indels, 150,344 short tandem repeats (STRs), 3,425 structural variants (SVs), and 2,826 differentially expressed genes (DE genes) among these 14 strains; 312,981 SNPs (89%) distinguished the B6 and B10 lineages. These SNPs were clustered into 28 short segments that are likely due to introgressed haplotypes rather than new mutations. Outside of these introgressed regions, we identified 53 SVs, protein-truncating SNPs, and frameshifting indels that were associated with DE genes. Our results can be used for both forward and reverse genetic approaches and illustrate how introgression and mutational processes give rise to differences among these widely used inbred substrains.

Nonsense-mediated RNA decay: an emerging modulator of malignancy.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that selectively degrades RNAs harbouring truncating mutations that prematurely terminate translation, including nonsense, frameshift and some splice-site mutations. Recent studies show that NMD shapes the mutational landscape of tumours by selecting for mutations that tend to downregulate the expression of tumour suppressor genes but not oncogenes. This suggests that NMD can benefit tumours, a notion further supported by the finding that mRNAs encoding immunogenic neoantigen peptides are typically targeted for decay by NMD. Together, this raises the possibility that NMD-inhibitory therapy could be of therapeutic benefit against many tumour types, including those with a high load of neoantigen-generating mutations. Complicating this scenario is the evidence that NMD can also be detrimental for many tumour types, and consequently tumours often have perturbed NMD. NMD may suppress tumour generation and progression by degrading subsets of specific normal mRNAs, including those encoding stress-response proteins, signalling factors and other proteins beneficial for tumours, as well as pro-tumour non-coding RNAs. Together, these findings suggest that NMD-modulatory therapy has the potential to provide widespread therapeutic benefit against diverse tumour types. However, whether NMD should be stimulated or repressed requires careful analysis of the tumour to be treated.

Very late onset LASIK flap Acremonium fungal keratitis confirmed by metagenomic deep sequencing.

PURPOSE: To describe a unique case of LASIK flap fungal keratitis confirmed by next generation sequencing. OBSERVATIONS: A 56-year-old female presented with refractory keratitis involving her LASIK flap 21 years after surgery. Confocal was positive for filamentous structures. The patient underwent immediate flap amputation followed by topical antifungal treatment. Corneal culture was positive for Acremonium sp. Metagenomic deep sequencing confirmed Acremonium as the primary source of infection and also identified Fusarium as a likely contributor of a mixed fungal infection. Sequencing also identified hay as the likely source of the infection. Treatment resulted in eradication of the infection. The patient's final best corrected visual acuity was 20/30 with rigid contact lens overrefraction. CONCLUSIONS: Metagenomic deep sequencing is a novel diagnostic tool that is increasingly being utilized for diagnosis of refractory keratitis. This case demonstrates the diagnostic potential of deep sequencing for identifying post-LASIK keratitis and reinforces the utility of LASIK flap amputation in the setting of tectonic flap instability due to keratolysis. IMPORTANCE: This case highlights several important clinical points for treating LASIK flap keratitis and highlights the emerging role metagenomic sequencing has in the diagnosis of infectious keratitis. This is first known case using next generation sequencing to diagnose a post-LASIK infectious keratitis.

Concordant Androgen-Regulated Expression of Divergent Rhox5 Promoters in Sertoli Cells.

Concordant transcriptional regulation can generate multiple gene products that collaborate to achieve a common goal. Here we report a case of concordant transcriptional regulation that instead drives a single protein to be produced in the same cell type from divergent promoters. This gene product-the RHOX5 homeobox transcription factor-is translated from 2 different mRNAs with different 5' untranslated regions (UTRs) transcribed from alternative promoters. Despite the fact that these 2 promoters-the proximal promoter (Pp) and the distal promoter (Pd)-exhibit different patterns of tissue-specific activity, share no obvious sequence identity, and depend on distinct transcription factors for expression, they exhibit a remarkably similar expression pattern in the testes. In particular, both depend on androgen signaling for expression in the testes, where they are specifically expressed in Sertoli cells and have a similar stage-specific expression pattern during the seminiferous epithelial cycle. We report evidence for 3 mechanisms that collaborate to drive concordant Pp/Pd expression. First, both promoters have an intrinsic ability to respond to androgen receptor and androgen. Second, the Pp acts as an enhancer to promote androgen-dependent transcription from the Pd. Third, Pd transcription is positively autoregulated by the RHOX5 protein, which is first produced developmentally from the Pp. Together, our data support a model in which the Rhox5 homeobox gene evolved multiple mechanisms to activate both of its promoters in Sertoli cells to produce Rhox5 in an androgen-dependent manner during different phases of spermatogenesis.

Optical coherence tomographic angiography study of perfusion recovery after surgical lowering of intraocular pressure.

We investigated the time and location of retinal perfusion recovery after surgical intraocular pressure (IOP) lowering in glaucoma by using optical coherent tomography angiography (OCTA). Seventeen patients were analyzed. The 4.5 × 4.5-mm OCTA scans centered on the disc were performed preoperatively and postoperatively at 6 weeks, 3 months, and 6 months. The peripapillary retinal nerve fiber layer (NFL) thickness, NFL plexus capillary density (NFLP-CD) and visual field (VF) were measured overall and in 8 corresponding sectors. The low-perfusion area (LPA) was used to assess the cumulative area where local NFLP-CD was significantly below normal. At 6 months, the average IOP decreased 5.3 mmHg (P = 0.004), LPA decreased by 15% (P = 0.005), and NFLP-CD improved by 12% (P < 0.001). The NFL thickness and VF mean deviation didn't change significantly at any time point. Among the sectors with significant preoperative NFLP-CD loss, the recovery at 6 months was greatest in sectors with minimal preoperative NFL thinning (P < 0.001). In conclusion, surgical IOP lowering may improve NFLP capillary perfusion after 6 months. The perfusion recovery tended to occur in areas with minimal NFL thinning at baseline. OCTA parameters may have potential usefulness as pharmacodynamic biomarkers for glaucoma therapy.