Functional mapping of PHF6 complexes in chromatin remodeling, replication dynamics, and DNA repair.

The Plant Homeodomain 6 gene (PHF6) encodes a nucleolar and chromatin-associated leukemia tumor suppressor with proposed roles in transcription regulation. However, specific molecular mechanisms controlled by PHF6 remain rudimentarily understood. Here we show that PHF6 engages multiple nucleosome remodeling protein complexes, including nucleosome remodeling and deacetylase, SWI/SNF and ISWI factors, the replication machinery and DNA repair proteins. Moreover, after DNA damage, PHF6 localizes to sites of DNA injury, and its loss impairs the resolution of DNA breaks, with consequent accumulation of single- and double-strand DNA lesions. Native chromatin immunoprecipitation sequencing analyses show that PHF6 specifically associates with difficult-to-replicate heterochromatin at satellite DNA regions enriched in histone H3 lysine 9 trimethyl marks, and single-molecule locus-specific analyses identify PHF6 as an important regulator of genomic stability at fragile sites. These results extend our understanding of the molecular mechanisms controlling hematopoietic stem cell homeostasis and leukemia transformation by placing PHF6 at the crossroads of chromatin remodeling, replicative fork dynamics, and DNA repair.

Development of an anti-SARS-CoV-2 monoclonal antibody panel and its applicability as a reagent in high-throughput fluorescence reduction neutralization and immunohistochemistry assays.

Since its emergence in late 2019, coronavirus disease 2019 (COVID-19) has caused millions of deaths and socioeconomic losses. Although vaccination significantly reduced disease mortality, it has been shown that protection wanes over time, and that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) may escape vaccine-derived immunity. Therefore, serological studies are necessary to assess protection in the population and guide vaccine regimens. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. We developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we developed and characterized monoclonal antibodies (mAbs) to lower costs and reduce the assay's vulnerability to reagent shortages. Using samples of individuals vaccinated with COVID-19 and unvaccinated/pre-pandemic samples, we showed that FRNA results using commercial and in-house mAbs strongly correlated with those of the PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high-throughput alternative for measuring nAbs, the FRNA can be easily customized to assess SARS-CoV-2 VOCs. Additionally, the mAb we produced was able to detect SARS-CoV-2 in pulmonary tissues by immunohistochemistry assays.

Cataract surgery and IOP: a systematic review of randomised controlled trials.

PURPOSE: Cataract and glaucoma are two of the most common ocular comorbidities. Cataract surgery has been shown to influence intra-ocular pressure (IOP) in patients with glaucoma; nevertheless, the extent of this effect remains controversial, especially in patients with open-angle glaucoma (OAG). The aim of this review is to determine the real effect of cataract surgery on IOP change in patients with OAG, focusing on data retrieved from randomised controlled trials (RCTs). METHODS: A systematic review was performed, including six different RCTs that studied the net effect of cataract surgery on IOP. Eligibility criteria required a full washout from hypotensive therapy, allowing accurate measurement of unmedicated IOP, both before and after surgery. RESULTS: Included studies revealed a consistent reduction on IOP occurring after surgery, varying between 4.1 and 8.5 mmHg depending on the RCT. There was also a decrease in the number of glaucoma medications, with a mean reduction of 0.2-1.0 agents postoperatively. Evaluation of adverse outcomes of cataract surgery showed a very favourable safety profile. CONCLUSION: Although the role of cataract surgery in the algorithm of glaucoma treatment remains to be established, this review highlights a consistent decrease on IOP following surgery and a reduced dependency on glaucoma medications. Potential downgrade in medication can thus be considered in well-controlled glaucoma patients after phacoemulsification. PROSPERO registry: CRD42022343378.

Effects of substratum type and orientation on the recruitment of bryozoans in an artificial area of the Western Atlantic.

Bryozoans are commonly associated with various artificial structures in marine environments and have been responsible for several bioinvasion events worldwide. Understanding the interactions between bryozoans and artificial structures is therefore essential to prevent the establishment and spread of potential bioinvaders. This study investigated bryozoan recruitment on four different substrates (PET, nautical ropes, metal, and PVC) placed in three orientations (vertical, horizontal facing down and facing up) in an area of the Western Atlantic. In total, 15 species of bryozoans were found. The results revealed significant variations in assemblages' richness, with bryozoans showing a preference for settling on PVC (14 species found) and on the underside of horizontal substrates (15 species found), resulting in the higher representativity observed in this study. Cryptogenic (nine species) and exotic (five species) bryozoans dominated the assemblages in all treatments, indicating that the type of substrate (especially artificial) and its orientation can favor the settlement of bryozoans, particularly non-native species. Therefore, the availability of multiple types of artificial substrates in marine environments should be treated as a cause for concern.

Quality of life in heterosexual menopausal women: The indirect effect of sexual and marital satisfaction, menopause representations, and psychological morbidity.

Menopause may negatively impact Quality of Life (QoL). Our study used a cross-sectional design and research participants were 99 women in natural menopause. In our study, we analyzed the relationship between age at menopause onset, hormone therapy use, duration of couple relationship, menopause duration, psychological morbidity, marital satisfaction, menopause representations, and QoL following the Wilson and Cleary Health-Related QoL conceptual model. The authors found that negative representations, lower marital satisfaction, psychological morbidity, and shorter duration of menopause contributed to lower QoL. Moreover, the authors found that psychological morbidity and menopause representations (identity and control/cure dimensions) had an indirect effect between marital satisfaction and vasomotor, psychosocial, and sexual QoL. Also, we found that age at the onset of menopause showed a moderating effect in the final model. Future studies should replicate these results in a longitudinal design and analyze how the variables that showed a moderating role and indirect effects will function as moderators and mediators, respectively, over time.

Metabolic control and quality of life in type 1 diabetes: Do adherence, family support, and school support matter?

This study analyzed the relationship between clinical and demographic variables, family support, school support, and adherence on adolescents' metabolic control and quality of life (QoL) based on Adaptation to Chronic Disease in Childhood's model. The sample included 100 adolescents and 100 parents. Adolescents were assessed on adherence, family support, school support, and QoL. Parents were assessed on family functioning and parental coping. A path analysis was performed to assess the adequacy of the theoretical model to the sample. Daily glycemic monitoring was associated with better metabolic control and diabetes hospitalizations with worse metabolic control. The final model showed adherence, family support, school support, and family functioning having an indirect effect on the relationship between adolescence stage, number of daily glycemic monitorings, number of hospitalizations, and metabolic control/QoL. Adherence had an indirect effect on the relationship between glycemic monitoring and family and school support, suggesting that adolescents need both types of support to perform diabetes self-care tasks. QoL showed a direct effect on metabolic control revealing the importance of QoL in the design of interventions to promote metabolic control in adolescents.

Quality of Life in Caregivers of Type 2 Diabetes Patients After Patient's Surgery: a Path Analysis.

BACKGROUND: Type 2 diabetes Mellitus is a chronic disease, and one of its complications may result in amputation. The goal of this study was to evaluate the impact of psychosocial variables, over time, on quality of life (QoL) of caregivers of patients (N = 110) who had undergone a recent amputation. METHOD: A longitudinal design was employed: (T1) 1 month after the patient's surgery; (T2) 7 months after; and (T3) 10 months after. Burden Assessment Scale, Family Assessment Device, Revised Impact of Events Scale, Family Disruption from Illness Scale, and Short Form Health Survey were used. A path analysis model was tested. RESULTS: Burden, perception of family functioning, and traumatic symptoms at T1 showed a positive impact on the perception of family functioning, at T2. Traumatic symptoms, at T1, predicted traumatic symptoms at T2 being also a mediator between those symptoms (T1) and physical QoL (T3). Physical and traumatic symptoms, at T1 predicted physical symptoms, at T2 affecting both mental and physical QoL (T3), being also a mediator in these relationships. Burden (T1) also had a direct effect on mental QoL (T3) and at T2 had an impact on physical QoL (T3) mediating also the relationship between burden/physical symptoms at T1 and physical QoL, at T3. CONCLUSION: Intervention should focus on physical and traumatic symptoms as well as burden since they were both mediators regarding mental and physical QoL.

The eye of the giant anteater (Myrmecophaga tridactyla): Ophthalmic examination findings and diagnostic tests.

OBJECTIVE: To describe ophthalmic examination and diagnostic values for Schirmer tear test (STT), intraocular pressure (IOP), corneal horizontal diameter (CHD), palpebral fissure length (PFL), fundoscopy, and palpebral conjunctiva microbiota from healthy giant anteaters. ANIMALS STUDIED: Twelve giant anteaters (Myrmecophaga tridactyla), 11 adults and one juvenile, five males and seven females. PROCEDURES: The animals were submitted to general anesthesia and ophthalmic evaluation with portable slit-lamp biomicroscope, Finoff transilluminator, and fundoscopy, as well as STT, bacterial culture from palpebral conjunctiva, rebound tonometry IOP, and measurement of PFL and CHD. Data compiled were analyzed with ANOVA and Tukey tests. RESULTS: The results (mean ± standard deviation) were as follows: STT 8.04 ± 6.21 mm/min; IOP 10.92 ± 2.45 mmHg; PFL 0.75 ± 0.11 cm; CHD 0.96 ± 0.10 cm. Out of the 24 eyes swab samples for bacterial culture, 17 were positive, with three genera of Gram-positive bacteria identified Staphylococcus spp., Bacillus sp., and Corynebacterium sp. Gram-negative bacteria were not isolated from any of the samples. CONCLUSIONS: As conservation work in this vulnerable species continues, this report on basic ophthalmic examination and diagnostic parameters will be helpful improve their treatment and care. More ophthalmic studies are encouraged in animals within the Pilosa order.

Ocular examination findings and measurements of tear production and tonometry of ring-tailed coatis (nasua nasua).

OBJECTIVE: To describe ocular anatomical features and to determine tear production and tonometry in normal anesthetized ring-tailed coati (Nasua nasua). ANIMALS: Eight male and seven female ring-tailed coatis (30 eyes) were evaluated, with ages ranging from 7 months to 10 years old. PROCEDURES: The animals were anesthetized with isoflurane and had STT 1, rebound and applanation tonometry, and palpebral fissure length (PFL) measured in both eyes. Rebound tonometry was measured with both TonoVet® and TonoVet Plus®. All eyes were evaluated with slit-lamp biomicroscopy. Following pharmacological mydriasis, direct fundoscopy was performed. ANOVA and Tukey tests were used for the data analysis. RESULTS: There were upper and lower eyelids, as well as a third eyelid. Eyelashes were present on the upper eyelid, while meibomian glands openings were visible on both. The bulbar conjunctiva was pigmented. The pupil was obliquely horizontally ovoid and teardrop shaped when miotic. The color of the iris and tapetum lucidum varied with age. The optic disk was round, and the retina was holangiotic. The mean ± standard deviation values obtained were as follows: STT: 2.50 ± 2.39 mm/min; rebound tonometry: 9.50 ± 2.43 mmHg (TonoVet®, calibration P), 14.60 ± 2.82 mmHg (TonoVet®, calibration D), 18.93 ± 3.99 mmHg (TonoVet Plus®, calibration dog); applanation tonometry: 18.66 ± 4.16 mmHg; PFL: 10.98 ± 1.67 mm. CONCLUSIONS AND CLINICAL RELEVANCE: Anatomical and clinical ophthalmic data presented in this study are unprecedented for this species. The results can be used as clinical reference parameters, allowing for better care of these species. The lack of data for the Procyonidae family must be regarded as an incentive for more studies.

Activating mutations and translocations in the guanine exchange factor VAV1 in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas frequently associated with poor prognosis and for which genetic mechanisms of transformation remain incompletely understood. Using RNA sequencing and targeted sequencing, here we identify a recurrent in-frame deletion (VAV1 Δ778-786) generated by a focal deletion-driven alternative splicing mechanism as well as novel VAV1 gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) in PTCL. Mechanistically these genetic lesions result in increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (nuclear factor of activated T cells, NFAT) VAV1 effector pathways. These results support a driver oncogenic role for VAV1 signaling in the pathogenesis of PTCL.

Influence of depth on bryozoan richness and distribution from the continental shelf of the northern coast of Bahia State, north-eastern Brazil.

Biological and abiotic processes influence ecosystem structure and species distribution. For bryozoan assemblages, depth, substratum and habitat structure are among the main factors influencing their distribution. Ecological studies on bryozoan fauna from Brazil are scarce and factors affecting the distribution and/or diversity of this common group are obscure. Here we attempted to verify the influence of bathymetry on bryozoan richness on the north shore of Bahia State, north-eastern Brazil. We identified 57 bryozoan taxa, comprising 35 families and 50 genera, all belonging to the Cheilostomata. Retevirgula multipunctata Winston, Vieira & Woollacott, 2014 and Tetraplaria dichotoma (Osburn, 1914) and the genus Aimulosia Jullien, 1888 were recorded for the first time from Bahia State. Species growing as encrusting sheets were dominant at all depths. There was a significant difference in richness among samples at different depths, with highest values of richness at 40 meters (Kruskal-Wallis Test). Analysis of similarities revealed a significant difference among the bryozoan assemblages, mainly when comparing assemblages from 10 and 20 meters with deeper ones. Also, shallower assemblages composition varied much than assemblages from 30 and 40 meters. Since depth is a proxy for several environmental parameters, further studies are needed to identify other factors influencing bryozoan distribution.

Ophthalmic contributions to assessing eyes of two neotropical canids: Cerdocyon thous and Chrysocyon brachyurus.

OBJECTIVE: To describe selected ophthalmic tests and anatomical features of eyes of crab-eating foxes (Cerdocyon thous) and maned wolves (Chrysocyon brachyurus). ANIMALS STUDIED: Six crab-eating foxes (12 eyes), eight maned wolves (16 eyes). PROCEDURES: Intramuscular and/or inhalatory anesthesia, ophthalmic evaluation with portable slit-lamp biomicroscope, Schirmer tear test (STT), intraocular pressure measurement with rebound and applanation (crab-eating fox only) tonometers, measurement of palpebral fissure length (PFL), gonioscopy, and fundoscopy. Data were analyzed with ANOVA and Tukey's tests. RESULTS: Both species presented upper and lower eyelids, both with eyelashes and Meibomian glands openings. A third eyelid was also present. In partial miosis, pigmented projections were observed along the edge of the pupil. The draining angle was open, with thin pectinate ligaments. The retina was holangiotic. For crab-eating foxes, mean ± standard deviation values were as follows: STT: 4.33 ± 2.96 mm/min; PFL: 17.45 ± 1.55 mm; rebound tonometry: 10.70 ± 3.43 mm Hg (TonoVet® calibration D),5.66 ± 3.44 mm Hg (TonoVet® calibration P), 17.00 ± 4.64 mm Hg (TonoVet® Plus calibration dog); and applanation tonometry: 11.70 ± 5.70 mm Hg(TonoPen® XL). For maned wolves, the mean ± standard deviation values were as follows: STT: 9.31 ± 7.40 mm/min; PFL: 22.79 ± 1.63 mm; rebound tonometry: 11.00 ± 2.77 mm Hg (TonoVet® calibration D), 6.78 ± 2.58 mm Hg (TonoVet® calibration P), and 18.29 ± 3.47 mm Hg(TonoVet® Plus calibration dog). CONCLUSIONS: This study contributes with knowledge that can help the clinical assessment regarding eyes of crab-eating foxes and maned wolves. The data herein presented for rebound tonometry are new for both species.

Adherence and Glycemic Control in Adolescents with Type 1 Diabetes: The Moderating Role of Age, Gender, and Family Support.

Adherence and glycemic control usually decrease during adolescence and family relationships influence diabetes outcomes. This study analyzed the interaction effect of adolescents' family support, age, and gender in the relationship between adherence and glycemic control in adolescents with Type 1 Diabetes. The sample included 100 adolescents with Type 1 Diabetes and one of their parents during a routine endocrinology appointment. Adolescents answered the Self-Care Inventory-Revised, the Diabetes Family Behavior Scale and were also assessed on the glycosylated hemoglobin. The three-way interaction between adherence, family support, and adolescents' age/gender was both negatively significant and explained 24.12% and 22.02% of the variance, respectively. Higher family support, being female, and younger age moderated the relationship between adherence and glycemic control. According to results, it is important that intervention programs provide negotiation skills, according to adolescent's age and gender in the process of transferring diabetes management responsibility in order to promote better adherence to diabetes self-care, glycemic control, and prevent family conflicts regarding diabetes management.

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam.

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.

Malassezia colonisation on a reconstructed human epidermis: Imaging studies.

BACKGROUND: Biofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. OBJECTIVE: The present study aimed to evaluate Malassezia colonisation pattern on a reconstructed human epidermis (RhE) by imaging techniques. METHODS: Malassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrhoeic dermatitis. Yeast of two strains of M furfur and M sympodialis were inoculated onto the SkinEthic™ RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning electron microscopy. RESULTS: Colonisation of the RhE surface with aggregates of Malassezia yeast entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeast were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded. Slight differences were found in the biofilm architectural structure between the two tested species with an apparently higher entrapment and viscosity in M furfur biofilm. CONCLUSION: Skin isolates of M furfur and M sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found.

Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy.

The response of aggregated Pseudomonas putida CP1 cells to UV-C and UV-A/B disinfection.

UV radiation is a spread method used worldwide for the disinfection of water. However, much of the research on the disinfection of bacterial cells by UV has focused on planktonic cells. Many bacterial cells in nature are present in clumps or aggregates, and these aggregates, which are more resistant to disinfection than their planktonic counterparts, can be problematic in engineered water systems. The current research used Pseudomonas putida (P. putida) CP1, an environmental and non-pathogenic microorganism which autoaggregates when grown under certain conditions, as a model organism to simulate aggregated cells. The study investigated the response of both the planktonic and the aggregated forms of the bacterium to UV-C (λ = 253.7 nm) and UV-A/B (λ > 300 nm) disinfection at laboratory scale in a minimal medium. The planktonic cells of P. putida CP1 were inactivated within 60 s by UV-C and in 60 min by UV-A/B; however, the aggregated cells required 120 min of UV-C treatment and 240 min of UV-A/B radiation to become inactive. The size of the aggregate was reduced following UV treatment. Although all the cells had lost culturability, viability as measured by the LIVE/DEAD® stain and epifluorescence microscopy was not completely lost and the cells all demonstrated regrowth after overnight incubation in the dark.

ßTrCP interacts with the ubiquitin-dependent endocytosis motif of the GH receptor in an unconventional manner.

GH (growth hormone) binding to the GHR (GH receptor) triggers essential signalling pathways that promote growth and metabolic regulation. The sensitivity of the cells to GH is mainly controlled by the endocytosis of the receptor via ßTrCP (ß-transducin repeat-containing protein). In the present study, we show that ßTrCP interacts directly via its WD40 domain with the UbE (ubiquitin-dependent endocytosis) motif in GHR, promoting GHR ubiquitination in vitro. NMR experiments demonstrated that the UbE motif is essentially unstructured, and, together with functional mapping of the UbE and ßTrCP WD40 residues necessary for binding, led to a unique interaction model of ßTrCP with GHR-UbE. This interaction is different from the conventional ßTrCP-substrate interactions described to date. This interaction therefore represents a promising specific target to develop drugs that inhibit GHR endocytosis and increase GH sensitivity in cachexia patients.

Ubc13 and COOH terminus of Hsp70-interacting protein (CHIP) are required for growth hormone receptor endocytosis.

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCF(ßTrCP) (Skp Cullin F-box). Despite a specific interaction between ß-transducin repeat-containing protein (ßTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCF(ßTrCP)-directed Lys(48) polyubiquitination. We now show that also Lys(63)-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to ßTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR.