Transcription Factors behind MYB98 Regulation: What Does the Discovery of SaeM Suggest?

MYB98 is master regulator of the molecular network involved in pollen tube attraction. Until recently, it was unclear how this gene exhibits exclusively synergid cell-specific expression in ovule. Our recent study has established that a 16-bp-long SaeM element is crucial for its synergid cell-specific expression in ovule, and an 84-bp-long fragment harboring SaeM is sufficient to drive the process. In this study, we have developed a workflow to predict functional roles of potential transcription factors (TFs) putatively binding to the promoter region, taking MYB98 promoter as a test subject. After sequential assessment of co-expression pattern, network analysis, and potential master regulator identification, we have proposed a multi-TF model for MYB98 regulation. Our study suggests that ANL2, GT-1, and their respective homologs could be direct regulators of MYB98 and indicates that TCP15, TCP16, FRS9, and HB34 are likely master regulators of the majority of the TFs involved in its regulation. Comprehensive studies in the future are expected to offer more insights into such propositions. Developed workflow can be used while designing similar regulome-related studies for any other species and genes.

Evolutionary assessment of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in citrus relatives with a specific focus on flowering.

Phase transition and floral induction in citrus requires several years of juvenility after germination. Such a long period of juvenility has been a major hindrance to its genetic improvement program. Studies have shown that miR156 along with its downstream genes SQUAMOSA PROMOTER BINDING PROTEINS (SBP) and SBP-LIKE (SPL) mediate the phase transition and floral induction process in plants. Our current study has systematically analyzed SPLs in 15 different citrus-related species, systematically annotated them based on their close homology to their respective Arabidopsis orthologs, and confirmed the functional attributes of the selected members in floral precocity. The majority of the species harbored 15 SPLs. Their cis-element assessment suggested the involvement of the SPLs in diverse developmental and physiological processes in response to different biotic and abiotic cues. Among all, SPL5, SPL9, and SPL11 stood out as consistently differentially expressed SPLs in the adult and young tissues of different citrus-related species. Independent overexpression of their F. hindsii orthologs (FhSPL5, FhSPL9, and FhSPL11) brought an enhanced expression of endogenous FLOWERING LOCUS T leading to the significantly precocious flowering in transgenic Arabidopsis lines. Future study of the genes in the citrus plant itself is expected to conclude the assessments made in the current study.

Discovery of a cis-regulatory element SaeM involved in dynamic regulation of synergid-specific MYB98.

MYB98 is a key regulator of the genetic network behind pollen tube attraction toward the female gametophyte. MYB98 is specifically expressed in the synergid cells (SCs), a female gametophyte component cells specialized for pollen tube attraction. However, it had not been clear how exactly MYB98 achieves this specific expression pattern. In the current study, we have determined that a normal SC-specific expression of MYB98 is dependent on a 16-bp-long cis-regulatory element, CATTTACACATTAAAA, freshly named as the "S ynergid-specific A ctivation E lement of M YB98" (SaeM). An 84 bp fragment harboring SaeM in the middle was sufficient to drive exclusively SC-specific expression. The element was present in a significantly large proportion of SC-specific gene promoters and in the promoter of MYB98 homologous genes in the Brassicaceae (pMYB98s). Significance of such family-wide SaeM-like element conservation in exclusive SC-specific expression was confirmed by the Arabidopsis-like activation feature of Brassica oleracea-derived pMYB98 and absence of such feature of pMYB98 derived from a non-Brassicaceae member Prunus persica. Additionally, the yeast-one-hybrid assay showed that the SaeM can be recognized by ANTHOCYANINLESS2 (ANL2) and DAP-seq data further suggested for additional three ANL2 homologs targeting the similar cis-element. Overall, our study has concluded that SaeM plays a crucial role in driving exclusively SC-specific expression of MYB98 and strongly suggests for the involvement of ANL2 and its homologs in its dynamic regulation in planta. Future study on the transcription factors is expected to shed more light on the mechanism behind the process.

Protocol for isolation of melanopsin and rhodopsin in the human eye using silent substitution.

Melanopsin-mediated visual and non-visual functions are difficult to study in vivo. To isolate melanopsin responses, non-standard light stimulation instruments are required, with at least as many primaries as photoreceptor classes in the eye. In this protocol, we describe the physical light calibrations of the display instrumentation, control of stimulus artefacts, and correction of individual between-eye differences in human observers. The protocol achieves complete photoreceptor silent substitution in psychophysical, pupillometry, and electroretinographic experiments for probing melanopsin, rod, and cone function. For complete details on the use and execution of this protocol, please refer to Uprety et al. (2022).1.

Granulomatosis With Polyangiitis Presenting With Diffuse Alveolar Hemorrhage: A Systematic Review.

Granulomatosis with polyangiitis (GPA) is a systemic disease that has variable clinical expression. GPA is the most common antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Diffuse alveolar hemorrhage (DAH) is one of the least common pulmonary manifestations in patients with GPA. DAH is clinically marked by hemoptysis, anemia, and diffuse alveolar infiltrates on imaging as well as hypoxemic respiratory failure. The diagnosis and treatment are challenging. Recommendations for ANCA-associated vasculitis, in general, are already established; however, there is a knowledge gap that addresses the association of GPA and DAH. The aim of this systematic review is to focus on the main clinical aspects and treatment of patients with GPA who present with DAH. Thorough research of available literature was performed, including studies published in the last 10 years, following the Preferred Items for Systematic Review and Meta-Analysis (PRISMA) 2020 recommendations. The following databases were included: PubMed, Medline, Embase, ClinicalTrials.com, Google Scholar, and Prospero. The search terms included: [granulomatosis] AND [polyangiitis] AND [diffuse alveolar hemorrhage] OR [diffuse pulmonary hemorrhage] NOT [microscopic polyangiitis] NOT [eosinophilic granulomatosis]. NOS was used to assess the internal validity of the study in four domains, including selection, ascertainment, causality, and reporting. Our initial search identified 8989 studies. After excluding duplicated data and using our predetermined inclusion and exclusion criteria, we were able to retrieve 18 studies. Twelve out of 18 (67%) studies were case reports. Five were retrospective cohorts and one controlled trial. The average age of patients with GPA with DAH was 49.55 ± 17.54 years (18 - 76). Male individuals had a slight predominance (59%) in comparison to female individuals (41%). The hemoglobin level at the time of presentation was 8.86 mg/dL ± 1.43. The majority of patients (61.5%) reported hemoptysis. Renal involvement was present in 66.7%. Patients who required mechanical ventilation represented 61.5%. Plasmapheresis was used in 71.4%. Mortality was 20%, and gender was not associated with mortality (p = 0.822). Hemoptysis was not associated with the need for mechanical ventilation (p = 0.928). Renal impairment was not a predictor of mechanical ventilation (p = 0.207). In summary, patients with GPA and DAH were severely ill, frequently had renal impairment upon admission, and frequently required mechanical ventilation. Steroids, rituximab, and cyclophosphamide are the first-line treatment, and plasmapheresis is still in use. Eventually, extracorporeal membrane oxygenation (ECMO) can be the salvage therapy. Randomized controlled clinical trials (RCTs) are needed to address the best therapeutic options for this population.

The high-quality genome of pummelo provides insights into the tissue-specific regulation of citric acid and anthocyanin during domestication.

Citric acid and anthocyanin contents were co-selected during Citrus domestication. Pummelo is a founding species in the Citrus genus, but the domestication of pummelo has not been well studied. Here, we compared the citric acid and anthocyanin contents of a low citric acid pummelo (Citrus maxima LCA) and its high citric acid variety (HCA) from the same cultivation area in China. Our study revealed that, unlike the LCA type, the HCA variety accumulated anthocyanin in the pericarp early in fruit development. To investigate the genetic basis of acid and anthocyanin enrichment in HCA pulp and pericarp, respectively, we generated a chromosome-scale HCA genome using long-read sequence reads and Hi-C sequencing data. Transcriptome analysis and transient overexpression assays showed that the accumulation of citric acid and anthocyanin was associated with high expression of CgANTHOCYANIN1 (CgAN1), and two different MYBs transcription factors (CgPH4 and CgRuby1), respectively. Moreover, the CgAN1 promoter was more methylated in the LCA pulp than in the HCA pulp. Treatment with a DNA methylation inhibitor, 5-azacytidine, alleviated the CgAN1 promoter hypermethylation in the LCA pulp, leading to increased CgAN1 expression and citric acid content. This study provides a new high-quality pummelo genome and insight into the molecular mechanism behind the change in tissue-specific citric acid and anthocyanin accumulation during pummelo domestication and provides a conceptual basis for precise genetic manipulation in fruit flavor breeding.

A Rare Case of Pyriform Sinus Fistula in an Adult.

The pyriform sinus fistula is very rare in adults, and it can present as recurrent deep neck space infection and abscess formation. The fistula results due to failure of obliteration of the third and fourth pharyngeal pouches. The diagnosis is often challenging, even with standard imaging techniques. We present a case of a patient who developed recurrent neck space infection and abscess formation. She was found to have a pyriform sinus fistula with fluoroscopy esophagram, although it was not seen with a standard contrast-enhanced computed tomography scan of the neck.

Photonics probing of pup brain tissue and molecular-specific nuclear nanostructure alterations due to fetal alcoholism via light scattering/localization approaches.

SIGNIFICANCE: Light is a good probe for studying the nanoscale-level structural or molecular-specific structural properties of brain cells/tissue due to stress, alcohol, or any other abnormalities. Chronic alcoholism during pregnancy, i.e., fetal alcoholism, being teratogenic, results in fetal alcohol syndrome, and other neurological disorders. Understanding the nano-to-submicron scale spatial structural properties of pup brain cells/tissues using light/photonic probes could provide a plethora of information in understanding the effects of fetal alcoholism. AIM: Using both light scattering and light localization techniques to probe alterations in nano- to-submicron scale mass density or refractive index fluctuations in brain cells/tissues of mice pups, exposed to fetal alcoholism. APPROACH: We use the mesoscopic physics-based dual spectroscopic imaging techniques, partial wave spectroscopy (PWS) and molecular-specific inverse participation ratio (IPR) using confocal imaging, to quantify structural alterations in brain tissues and chromatin/histone in brain cells, respectively, in 60 days postnatal mice pup brain, exposed to fetal alcoholism. RESULTS: The finer focusing PWS analysis on tissues shows an increase in the degree of structural disorder strength in the pup brain tissues. Furthermore, results of the molecular-specific light localization IPR technique show an increase in the degree of spatial molecular mass density structural disorder in DNA and a decrease in the degree in histone. CONCLUSIONS: In particular, we characterize the spatial pup brain structures from the molecular to tissue levels and address the plausible reasons for such as mass density fluctuations in fetal alcoholism.

Melanopsin photoreception differentially modulates rod-mediated and cone-mediated human temporal vision.

To evaluate the nature of interactions between visual pathways transmitting the slower melanopsin and faster rod and cone signals, we implement a temporal phase summation paradigm in human observers using photoreceptor-directed stimuli. We show that melanopsin stimulation interacts with and alters both rod-mediated and cone-mediated vision regardless of whether it is perceptually visible or not. Melanopsin-rod interactions result in either inhibitory or facilitatory summation depending on the temporal frequency and photoreceptor pathway contrast sensitivity. Moreover, by isolating rod vision, we reveal a bipartite intensity response property of the rod pathway in photopic lighting that extends its operational range at lower frequencies to beyond its classic saturation limits but at the expense of attenuating sensitivity at higher frequencies. In comparison, melanopsin-cone interactions always lead to facilitation. These interactions can be described by linear or probability summations and potentially involve multiple intraretinal and visual cortical pathways to set human visual contrast sensitivity.

Genome-wide identification of ovate family in Citrus and functional characterization of CitOFP19.

Fruit shape is an important trait for fruit appearance and commercial value. Diversity of fruit-shape has been utilized in the breeding of pummelo (Citrus maxima), a basic species in Citrus. However, little is known about genetic basis of fruit shape in citrus. In this study, we identified 16 OVATE family protein (OFP) genes in the pummelo genome. Phylogenetically, they were classified into three subfamilies, which was consistent with the classification of their Arabidopsis orthologs. Synteny analysis suggested that segment and tandem duplications were responsible for their expansion in pummelo. Expression pattern analysis of Citrus OFPs (CitOFPs) showed that CitOFP19 had significantly higher expression level in the ovaries of round pummelo than in those of pear-shaped pummelo. Heterologous overexpression of CitOFP19 in tomato resulted in pear-shaped ovary and fruit shape. Taken together, this study characterized OVATE gene family in Citrus genome and assessed the function of CitOFP19.

Sepsis Due to Haemophilus influenzae and Masked Influenza A Infection.

Haemophilus influenzae (H. influenzae) is a facultative anaerobe, pleomorphic Gram-negative coccobacillus capable of causing various respiratory and blood stream infections. Introduction of childhood immunization against H. influenza type b has decreased its prevalence. Invasive infection with non-typeable H. influenzae is increasing specially in vulnerable population. We present a case of a 69-year-old female who developed septic shock due to H. influenzae infection. She was also found to have influenza A infection in bronchoalveolar lavage (BAL) sample although initial test with nasopharyngeal swab was negative. This case report highlights the fact that in patients with high clinical suspicion, negative nasopharyngeal swab with polymerase chain reaction may not rule out influenza infection and BAL may be necessary to confirm the diagnosis and H. influenzae can be causing bacterial superinfection in such patients. She was appropriately treated with third-generation cephalosporin for H. influenzae and with oseltamivir for influenza A. Her condition improved significantly with the treatment.

The role of melanopsin photoreception on visual attention linked pupil responses.

A decision during a visual task is marked by a task-evoked pupil dilation (TEPD) that is linked to the global cortical arousal state. Melanopsin expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) form the afferent pathway for this pupil response. Melanopsin activation also influences mood and arousal and increases activity in decision-making brain areas that receive direct ipRGC projections. Here, an optical photostimulation method controlled the excitations of all five photoreceptor classes in the human eye to isolate melanopsin-mediated photoreception. We hypothesised that the TEPD can be driven by directing active visual covert attention through the ipRGC pathway. When observers are completely certain of the stimulus presence, melanopsin-directed stimulation produces a TEPD of similar amplitude to a cone-directed stimulation, with their combination producing larger amplitudes. This dilation is satisfactorily modelled by linear addition with a higher melanopsin weighting in ipRGCs. Visual reaction times were longest in response to melanopsin-directed lights. Next, we asked whether the afferent photoreceptor input and decision certainty, controlled by priming the observer's a priori expectation, interact to drive the TEPD. Signal detection analysis showed that by fixing the predecision certainty (bias), the phasic arousal and TEPD amplitude vary with observer criterion (c') and sensitivity (d') but not with preferential activation of melanopsin. The signature feature of the melanopsin response during attention was a biphasic TEPD. We conclude that active covert attention can be modulated by visual information mediated via ipRGCs, but that phasic arousal responses marked using the TEPD are not increased by higher levels of melanopsin activation.

Multi-component redox system for selective and potent antineoplastic activity towards ovarian cancer cells.

Ovarian cancer is the deadliest gynecological cancer which rarely causes symptoms, and goes undetected until reaching the advanced stage of drug-resistant metastases. The cationic porphyrin meso-tetra(4-N-methylpyridyl)porphine (TMPyP) is a well-known photosensitizer (PS) used in photodyamic therapy (PDT) for curing cancer due to its strong affinity for DNA and high yield of reactive oxygen species (ROS) upon light activation. The practicality to irradiate tumor cells alone in the physiological system being slim (due to the close proximity of healthy cells and tumors), we looked for a variation in the PDT using a mixture of TMPyP with 1,5-dihydroxynapthalene (DHN) and Fe(III) ions at a mole ratio of 1:20:17 (drug combo) respectively in aqueous solution. The drug combo needs no photoactivation in H2O2 rich environment (mimicking the microenvironment of cancer/tumor), where it generates È®H and juglone, the latter being a known potent anticancer agent. In vitro studies of the drug combo in drug resistant and sensitive ovarian cancer cell lines showed drastic growth inhibition and cell death compared to normal epithelial cells. The drug combo provides an effective and non-invasive alternative to conventional PDT, exploiting the cytosolic carcinogenic H2O2 to produce an efficient anticancer treatment. The unique action of cancer-specific cytotoxicity arises from the redox chemistry involving activation of Fe(III) as the oxidizing agent to generate juglone, which utilizes the cytosolic ROS in cancer cells against itself.

Design and validation of a chart-based measure of the limits of spatial contrast sensitivity.

PURPOSE: Current chart-based tests of spatial contrast sensitivity (SCS) with fixed or narrow frequency ranges (≤18 cycles/°) cannot characterise the limits of spatial contrast vision. Here we present the design and validation of a chart-based measure of the spatial contrast envelope. METHODS: Following the principles of the standard visual acuity (Bailey-Lovie) and contrast sensitivity (Pelli-Robson) charts, a combined spatial-contrast and visual acuity chart was designed using a language-independent triangular symbol for a four-alternative forced-choice procedure plus chart rotation. Symbol frequencies ranged between 0.38 and 60 cycles/° spaced along 10 radial axes (0.55%-100% contrast). The chart was validated with reference to the Bailey-Lovie and Pelli-Robson charts; its reliability and sensitivity to changes in illumination, simulated cataract and blur was evaluated in healthy adults. RESULTS: The photopic SCS function could be measured in 5.5 ± 0.5 min; thresholding around the spatial contrast resolution limit reduced completion times to ~2 min. There was good agreement with high-contrast visual acuity (difference = 0.08 ± 0.02 logMAR) and contrast-sensitivity at 1.5 cycles/° (0.13 ± 0.06 logCS). Test-retest reliability was excellent at all spatial frequencies (ICC = 0.99). Mesopic illumination or simulated cataract caused a generalised SCS loss; myopic blur reduced high-frequency sensitivity. Spatial contrast sensitivity was independent of radial axis orientation (cardinal or oblique). CONCLUSIONS: The chart provides a time-efficient, reliable and inexpensive measure of SCS with applications in research and clinic for detecting subtle deficits in early stages of ocular and neurological conditions that often manifest at higher frequencies. It is sensitive to vision changes occurring in dim lighting and with simulated cataract and blur. The chart is available open-access for self-printing; contrast variation in print can be controlled through user calibration and/or establishing normative SCS functions using the theoretical values.

Photonic technique to study the effects of probiotics on chronic alcoholic brain cells by quantifying their molecular specific structural alterations via confocal imaging.

Molecular specific photonics localization method, the inverse participation ratio (IPR) technique, is a powerful procedure to probe the nano- to submicron scales structural alterations in cells/tissues in their abnormalities due to chronic alcoholism using confocal imaging. Chronic alcoholism introduces abnormalities in brain cells/tissue at the nanoscale level that results in behavioural and psychological disorders which are not well understood. On the other hand, probiotics such as Lactobacillus plantarum enhances brain functions in chronic alcoholism. Using the IPR technique, we probe the molecular specific spatial structural alterations in glial brain cells astrocytes and microglia, as well as in chromatins in the nuclei of cortex brain cells, with or without probiotic treatments in chronic alcoholism. The results show chronic alcoholism alone harms brain cells and the probiotic treatment in chronic alcoholism reverses alcoholic damage in the brain cells/tissues toward normalcy.

Optimizing methods to isolate melanopsin-directed responses.

The intrinsic melanopsin photoresponse may initiate visual signals that differ in spatiotemporal characteristics from the cone-opsin- and rhodopsin-mediated signals. Applying the CIE standard observer functions in silent-substitution methods can require individual differences in photoreceptor spectral sensitivities and pre-receptoral filtering to be corrected; failure to do so can lead to the intrusion of more sensitive cone processes with putative melanopsin-directed stimuli. Here we evaluate heterochromatic flicker photometry (HFP) and photoreceptor-directed temporal white noise as techniques to limit the effect of these individual differences. Individualized luminous efficiency functions (V(λ)) were compared to the CIE standard observer functions. We show that adapting chromaticities used in silent-substitution methods can deviate by up to 54% in luminance when estimated with the individual and standard observer functions. These deviations lead to inadvertent cone intrusions in the visual functions measured with melanopsin-directed stimuli. To eliminate the intrusions, individual HFP corrections are sufficient at low frequencies (∼1Hz) but temporal white noise is also required at higher frequencies to desensitize penumbral cones. We therefore recommend the selective application of individualized observer calibration and/or temporal white noise in silent-substitution paradigms when studying melanopsin-directed photoresponses.

Light adaptation characteristics of melanopsin.

Following photopigment bleaching, the rhodopsin and cone-opsins show a characteristic exponential regeneration in the dark with a photocycle dependent on the retinal pigment epithelium. Melanopsin pigment regeneration in animal models requires different pathways to rods and cones. To quantify melanopsin-mediated light adaptation in humans, we first estimated its photopigment regeneration kinetics through the photo-bleach recovery of the intrinsic melanopsin pupil light response (PLR). An intense broadband light (~120,000 Td) bleached 43% of melanopsin compared to 86% of the cone-opsins. Recovery from a 43% bleach was 3.4X slower for the melanopsin than cone-opsin. Post-bleach melanopsin regeneration followed an exponential growth with a 2.5 min time-constant (τ) that required 11.2 min for complete recovery; the half-bleaching level (Ip) was ~ 4.47 log melanopic Td (16.10 log melanopsin effective photons.cm-2.s-1; 8.25 log photoisomerisations.photoreceptor-1.s-1). The effect on the cone-directed PLR of the level of the melanopsin excitation during continuous light adaptation was then determined. We observed that cone-directed pupil constriction amplitudes increased by ~ 10% when adapting lights had a higher melanopic excitation but the same mean photometric luminance. Our findings suggest that melanopsin light adaptation enhances cone signalling along the non-visual retina-brain axis. Parameters τ and Ip will allow estimation of the level of melanopsin bleaching in any light units; the data have implications for quantifying the relative contributions of putative melanopsin pathways to regulate the post-bleach photopigment regeneration and adaptation.

A Rare Case of Subacute Sclerosing Panencephalitis Presenting As Generalized Seizure.

Subacute sclerosing panencephalitis (SSPE) is a late complication of childhood measles. It is characterized by a progressive decline in cognitive and motor functions, seizures, and eventually death. Although a combination of intrathecal interferon alpha (IFN-α) and daily oral isoprinosine has been reported to have a good outcome, there is no cure for this condition.  We present a case of a 16-year-old male with SSPE who presented with progressive weakness, frequent loss of postural control, multiple episodes of generalized tonic-clonic seizures, and urinary incontinence. On exploration of his history, he had measles at the age of two months. Investigation showed increased serum measles antibody titer, high amplitude spikes in electroencephalogram (EEG), and high fluid-attenuated inversion recovery (FLAIR) signals on MRI of the brain consistent with probable SSPE. He was managed symptomatically until his condition got worse and he eventually passed away.

Association Between Myasthenia Gravis and Systemic Lupus Erythematosus as a Comorbid State.

Systemic lupus erythematosus (SLE) and myasthenia gravis (MG) are autoimmune states which have presentational similitude. Both conditions test serologically positive for anti-nuclear antibodies and require exceptional differential diagnostic acumen to segregate one from the other. The hypothesized factors provoking these diseases may be immunological, genetic, hormonal, or environmental and can be better understood by large-scale controlled epidemiological studies. Biochemical factors such as variation in CXC (an α chemokine subfamily), CXCL13, and granulocyte-macrophage colony-stimulating factor levels are assumed to play a pivotal role in the pathogenesis of SLE and MG; however, further studies are required to understand their exact mechanism and effect on the underlying autoimmune diseases. Following this, another precipitating factor for this overlap is believed to be thymectomy which is performed to eliminate MG symptoms. Although thymectomy is the effective treatment modality in MG patients, other findings and data support the view that this procedure may lead to the development of other autoimmune states such as SLE. It is evident from previously published data and case reports that patients with one autoimmune disease who underwent thymectomy contracted SLE and became more susceptible to other autoimmune diseases compared to the general population. Post-thymectomy follow-up of patients provides us with mechanistic clues for understanding the development of SLE-MG overlap; hence, in MG patients who have undergone thymectomy, any clinical and immune serological SLE suspicion should be carefully evaluated.