Comprehensive proteomic profiling of early antral follicles from sheep.

The present study evaluates the proteome of early antral follicles from Ovis aries. Fifty follicles were collected from ovaries of adult ewes and extracted proteins were trypsin-digested, desalted and analyzed by LC-MS/MS. Genes were screened for potential modulation by miRNAs and protein data, subjected to functional enrichment analysis. Label-free mass spectrometry allowed the identification of 2503 follicle proteins, confirming vimentin, actin, lamin, heat shock proteins and histones as the most abundant ones. In silico analyses indicated that miRNAs modulate the expression of genes coding proteins of the sheep follicles involved in cell cycle, cell differentiation, aging, apoptosis, cell death, adipocyte differentiation, cell division. The most important biological processes associated with the follicle proteins were innate immune response, translation, adaptive immune response and protein folding, while molecular functions linked to the proteome of sheep antral follicles related to metal ion binding, ATP binding, oxygen binding, RNA binding and GTP binding, among others. Upload of 2503 Uniport accession codes through DAVID platform matched 1274 genes, associated with translation, metabolic process, proteolysis involved in cellular protein catabolic process, zona pellucida receptor complex and others. KEEG pathways analysis indicated genes correlated with ovine follicular development, with major pathways listed as carbon metabolism, biosynthesis of amino acids, glutathione metabolism, oxidative phosphorylation, fatty acid degradation and oocyte meiosis. This represents a comprehensive atlas of proteins expressed in sheep early antral follicles and will contribute to future identification of biomarkers for follicular development and oocyte maturation.

Corrigendum: ALG1-CDG Caused by Non-Functional Alternative Splicing Involving a Novel Pathogenic Complex Allele.

[This corrects the article DOI: 10.3389/fgene.2021.744884.].

ALG1-CDG Caused by Non-functional Alternative Splicing Involving a Novel Pathogenic Complex Allele.

This study reports on a Mexican mestizo patient with a multi-systemic syndrome including neurological involvement and a type I serum transferrin profile. Clinical exome sequencing revealed complex alleles in ALG1, the encoding gene for the chitobiosyldiphosphodolichol beta-mannosyltransferase that participates in the formation of the dolichol-pyrophosphate-GlcNAc2Man5, a lipid-linked glycan intermediate during N-glycan synthesis. The identified complex alleles were NM_019109.5(ALG1): c.[208 + 16_208 + 19dup; 208 + 25G > T] and NM_019109.5(ALG1): c.[208 + 16_208 + 19dup; 1312C > T]. Although both alleles carried the benign variant c.208 + 16_208 + 19dup, one allele carried a known ALG1 pathogenic variant (c.1312C > T), while the other carried a new uncharacterized variant (c.208 + 25G > T) causing non-functional alternative splicing that, in conjunction with the benign variant, defines the pathogenic protein effect (p.N70S_S71ins9). The presence in the patient's serum of the pathognomonic N-linked mannose-deprived tetrasaccharide marker for ALG1-CDG (Neu5Acα2,6Galß1,4-GlcNAcß1,4GlcNAc) further supported this diagnosis. This is the first report of an ALG1-CDG patient from Latin America.

Pituitary porcine FSH, and recombinant bovine and human FSH differentially affect growth and relative abundances of mRNA transcripts of preantral and early developing antral follicles in goats.

Three different sources of FSH (porcine pituitary, pFSH; recombinant bovine, rbFSH; and recombinant human, rhFSH) were compared during in vitro culture of preantral and early antral follicles of goats for 18 days. Treatments were: base medium supplemented with no FSH (control), 10, 50, or 100 mIU/mL pFSH (pFSH10, pFSH50, and pFSH100, respectively), 100 ng/mL rbFSH (rbFSH), and 50 mIU/mL rhFSH (rhFSH). There were evaluations of follicle morphology, antrum formation, growth rate, estradiol production, oocyte viability and chromatin configuration, and follicle wall relative abundance of mRNA transcript for MMP-9, TIMP-2, CYP17, CYP19A1, FSHR, Insulin-R, and BAX/BCL-2 ratio. Follicle degeneration rates were similar among all treatment groups at the end of culturing. When there were treatments with pFSH, however, there was a lesser (P < 0.05) percentage of intact follicles and estradiol production, and greater (P < 0.05) extrusion rates. Furthermore, with only pFSH10 (antral follicles) and pFSH100 (preantral and antral follicles) treatments, there was a lesser (P < 0.05) follicle growth. For preantral follicles, when there was addition of pFSH10, pFSH100, and rhFSH there was lesser (P < 0.05) oocyte meiotic resumption compared to control and rbFSH treatments. For antral follicles, when there were treatments with rhFSH and pFSH10 there was greater (P = 0.08 - P < 0.05) oocyte maturation. In conclusion, the source of FSH differentially affected gene expression, as indicated by mRNA abundances, and follicular dynamics of preantral and antral follicles in vitro. Addition of FSH during the in vitro culture improved the developmental outcomes only for antral follicles.

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue.

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.

Clinical and molecular findings in a cohort of ANO5-related myopathy.

OBJECTIVE: ANO5-related myopathy is an important cause of limb-girdle muscular dystrophy (LGMD) and hyperCKemia. The main descriptions have emerged from European cohorts, and the burden of the disease worldwide is unclear. We provide a detailed characterization of a large Brazilian cohort of ANO5 patients. METHODS: A national cross-sectional study was conducted to describe clinical, histopathological, radiological, and molecular features of patients carrying recessive variants in ANO5. Correlation of clinical and genetic characteristics with different phenotypes was studied. RESULTS: Thirty-seven patients from 34 nonrelated families with recessive mutations of ANO5 were identified. The most common phenotype was LGMD, observed in 25 (67.5%) patients, followed by pseudometabolic presentation in 7 (18.9%) patients, isolated asymptomatic hyperCKemia in 4 (10.8%) patients, and distal myopathy in a single patient. Nine patients presented axial involvement, including one patient with isolated axial weakness. The most affected muscles according to MRI were the semimembranosus and gastrocnemius, but paraspinal and abdominal muscles, when studied, were involved in most patients. Fourteen variants in ANO5 were identified, and the c.191dupA was present in 19 (56%) families. Sex, years of disease, and the presence of loss-of-function variants were not associated with specific phenotypes. INTERPRETATION: We present the largest series of anoctaminopathy outside Europe. The most common European founder mutation c.191dupA was very frequent in our population. Gender, disease duration, and genotype did not determine the phenotype.

In vitro culture of isolated preantral and antral follicles of goats using human recombinant FSH: Concentration-dependent and stage-specific effect.

The present study aimed to investigate a concentration-response curve of human recombinant FSH (hrFSH) for in vitro culture of isolated preantral and early antral follicles of goats. Isolated follicles were cultured for 18 days using the following treatments: basic culture medium (control); or control medium supplemented with 10, 50, and 100 mIU/mL of hrFSH. At the end of the culture, cumulus-oocyte complexes were recovered and subjected to in vitro maturation. The following endpoints were evaluated: follicle morphology, growth rate and antrum formation, oocyte viability and meiotic stage, and estradiol production, as well as relative expression of FSH receptor (FSHR), and steroidogenic enzyme (3ß-HSD, CYP17, and CYP19A1) genes. In antral follicles, the FSH addition at 50 mIU/mL increased follicular diameter and growth rate, percentage of fully developed oocytes, and oocyte diameter (P < 0.05), and tended to increase the percentage of MII oocytes when compared to the control (P = 0.07). With preantral follicles, FSH addition at 100 mIU/mL increased relative abundance of mRNA for CYP19A1 when compared to the control (P < 0.05). At the same FSH concentrations of 100 and 50 mIU/mL, there was a greater relatively abundance of mRNA for 3ß-HSD and CYP17 in preantral than in antral follicles (P < 0.05). For preantral and antral follicle comparisons when the same treatments were imposed, there were greater concentrations of estradiol for antral follicles (P < 0.05). In conclusion, hrFSH enhanced in a concentration-dependent manner the in vitro development of caprine antral follicles; however, there was no positive effect in the culture of preantral follicles.

Reference values for selected ophthalmic tests of the blue-and-yellow macaw (Ara ararauna) / Valores de referência para selecionados testes oftálmicos para a arara canindé (Ara araruna)

The aim of this study was to establish reference values for selected ophthalmic diagnostic tests in healthy blue-and-yellow macaws. We investigated a total of 35 adult macaws (70 eyes) of undetermined sex and with an average weight of 1 kg, who were living in captivity in the Federal District, Brazil. Tear production using the Schirmer tear test (STT), normal conjunctival flora, intraocular pressure (IOP) using a rebound tonometer and horizontal palpebral fissure length (HPFL) were evaluated. In this study, 84.1% of samples were positive for microbial growth. Bacteria, fungi and yeasts were isolated, and Staphylococcus spp. (21.9%) and Bacillus spp. (26.8%) were the most frequently isolated microorganisms. The mean value for STT was 7.6±4.6mm/min in the right eye (OD) and 6.6±4.4mm/min in the left eye (OS) (median = 7,11±0,76mm/min). Mean IOP was 11.4±2.5mm Hg OD and 11.6±1.8mm Hg OS (median = 11.49±0.22mm Hg), prior to anesthesia, and 7.6±2.4mm Hg OD and 7.8±1.8mm Hg OS (median 7.71±0.08mmHg) after anesthesia. The IOP was significantly lower when the animals were under anesthesia as compared to when they were conscious (p≤0.05). Horizontal palpebral fissure length was 11.7±0.1mm OD and 11.8±0.1mm OS (median = 11.72±0.07mm). The STT showed a positive correlation with palpebral fissure measurement for this species. These selected ophthalmic reference values will be particularly useful in diagnosing pathological changes in the eyes of blue-and-yellow macaws.(AU)

Objetivou-se determinar os valores normais para testes oftálmicos diagnósticos selecionados para a Arara Canindé. Trinta e cinco Ara ararauna (70 olhos), de sexo indeterminado, adultas, com peso médio de 1kg e provenientes de cativeiro no Distrito Federal, foram avaliadas. Aferiram-se a produção lacrimal pelo Teste lacrimal de Schirmer (TLS), a avaliação microbiológica da conjuntiva ocular, a pressão intra-ocular (PIO) utilizando a tonometria de rebote e o comprimento horizontal da rima palpebral Neste estudo 84.1% das amostras analisadas foram positivas para crescimento microbiológico. Bactérias, fungos e hifas foram isolados e Staphylococcus spp. (21.9%) e Bacillus spp. (26.8%) foram isolados mais frequentemente. Os valores médios do teste de Lacrimal de Schirmer (TLS) foram de 7.6±4.6 e 6.6±4.4mm/min para olhos direito (OD) e esquerdo (OE), respectivamente (média = 7,11±0,76mm/min). A pressão intraocular média foi de 11.4±2.5 (OD) e 11.6±1.8mmHg (OE) anteriormente à anestesia (média 11,49±0,22 mmHg) e 7.6 ± 2.4 mmHg (OD) e 7.8 ± 1.8 mm Hg (OE) (média 7,71±0,08mm Hg) após a anestesia, verificando-se que a PIO foi significativamente menor quando os animais se encontravam sob anestesia comparativamente ao momento em que não estavam anestesiados. O comprimento horizontal da rima horizontal palpebral foi de 11.7±0.1mm OD e de 11.8±0.1mm OE (média 11,72±0,07mm). Verificou-se correlação positiva do TLS com o comprimento da fissura palpebral para a espécie estudada. Estes valores de referencia serão úteis no diagnóstico de alterações oculares da Arara Canindé.(AU)

Reference values for selected ophthalmic tests of the blue-and-yellow macaw (Ara ararauna) / Valores de referência para selecionados testes oftálmicos para a arara canindé (Ara araruna)

The aim of this study was to establish reference values for selected ophthalmic diagnostic tests in healthy blue-and-yellow macaws. We investigated a total of 35 adult macaws (70 eyes) of undetermined sex and with an average weight of 1 kg, who were living in captivity in the Federal District, Brazil. Tear production using the Schirmer tear test (STT), normal conjunctival flora, intraocular pressure (IOP) using a rebound tonometer and horizontal palpebral fissure length (HPFL) were evaluated. In this study, 84.1% of samples were positive for microbial growth. Bacteria, fungi and yeasts were isolated, and Staphylococcus spp. (21.9%) and Bacillus spp. (26.8%) were the most frequently isolated microorganisms. The mean value for STT was 7.6±4.6mm/min in the right eye (OD) and 6.6±4.4mm/min in the left eye (OS) (median = 7,11±0,76mm/min). Mean IOP was 11.4±2.5mm Hg OD and 11.6±1.8mm Hg OS (median = 11.49±0.22mm Hg), prior to anesthesia, and 7.6±2.4mm Hg OD and 7.8±1.8mm Hg OS (median 7.71±0.08mmHg) after anesthesia. The IOP was significantly lower when the animals were under anesthesia as compared to when they were conscious (p≤0.05). Horizontal palpebral fissure length was 11.7±0.1mm OD and 11.8±0.1mm OS (median = 11.72±0.07mm). The STT showed a positive correlation with palpebral fissure measurement for this species. These selected ophthalmic reference values will be particularly useful in diagnosing pathological changes in the eyes of blue-and-yellow macaws.(AU)

Objetivou-se determinar os valores normais para testes oftálmicos diagnósticos selecionados para a Arara Canindé. Trinta e cinco Ara ararauna (70 olhos), de sexo indeterminado, adultas, com peso médio de 1kg e provenientes de cativeiro no Distrito Federal, foram avaliadas. Aferiram-se a produção lacrimal pelo Teste lacrimal de Schirmer (TLS), a avaliação microbiológica da conjuntiva ocular, a pressão intra-ocular (PIO) utilizando a tonometria de rebote e o comprimento horizontal da rima palpebral Neste estudo 84.1% das amostras analisadas foram positivas para crescimento microbiológico. Bactérias, fungos e hifas foram isolados e Staphylococcus spp. (21.9%) e Bacillus spp. (26.8%) foram isolados mais frequentemente. Os valores médios do teste de Lacrimal de Schirmer (TLS) foram de 7.6±4.6 e 6.6±4.4mm/min para olhos direito (OD) e esquerdo (OE), respectivamente (média = 7,11±0,76mm/min). A pressão intraocular média foi de 11.4±2.5 (OD) e 11.6±1.8mmHg (OE) anteriormente à anestesia (média 11,49±0,22 mmHg) e 7.6 ± 2.4 mmHg (OD) e 7.8 ± 1.8 mm Hg (OE) (média 7,71±0,08mm Hg) após a anestesia, verificando-se que a PIO foi significativamente menor quando os animais se encontravam sob anestesia comparativamente ao momento em que não estavam anestesiados. O comprimento horizontal da rima horizontal palpebral foi de 11.7±0.1mm OD e de 11.8±0.1mm OE (média 11,72±0,07mm). Verificou-se correlação positiva do TLS com o comprimento da fissura palpebral para a espécie estudada. Estes valores de referencia serão úteis no diagnóstico de alterações oculares da Arara Canindé.(AU)

Reference values for selected ophthalmic tests of the blue-and-yellow macaw (Ara ararauna)

ABSTRACT: The aim of this study was to establish reference values for selected ophthalmic diagnostic tests in healthy blue-and-yellow macaws. We investigated a total of 35 adult macaws (70 eyes) of undetermined sex and with an average weight of 1 kg, who were living in captivity in the Federal District, Brazil. Tear production using the Schirmer tear test (STT), normal conjunctival flora, intraocular pressure (IOP) using a rebound tonometer and horizontal palpebral fissure length (HPFL) were evaluated. In this study, 84.1% of samples were positive for microbial growth. Bacteria, fungi and yeasts were isolated, and Staphylococcus spp. (21.9%) and Bacillus spp. (26.8%) were the most frequently isolated microorganisms. The mean value for STT was 7.6±4.6mm/min in the right eye (OD) and 6.6±4.4mm/min in the left eye (OS) (median = 7,11±0,76mm/min). Mean IOP was 11.4±2.5mm Hg OD and 11.6±1.8mm Hg OS (median = 11.49±0.22mm Hg), prior to anesthesia, and 7.6±2.4mm Hg OD and 7.8±1.8mm Hg OS (median 7.71±0.08mmHg) after anesthesia. The IOP was significantly lower when the animals were under anesthesia as compared to when they were conscious (p0.05). Horizontal palpebral fissure length was 11.7±0.1mm OD and 11.8±0.1mm OS (median = 11.72±0.07mm). The STT showed a positive correlation with palpebral fissure measurement for this species. These selected ophthalmic reference values will be particularly useful in diagnosing pathological changes in the eyes of blue-and-yellow macaws.

RESUMO: Objetivou-se determinar os valores normais para testes oftálmicos diagnósticos selecionados para a Arara Canindé. Trinta e cinco Ara ararauna (70 olhos), de sexo indeterminado, adultas, com peso médio de 1kg e provenientes de cativeiro no Distrito Federal, foram avaliadas. Aferiram-se a produção lacrimal pelo Teste lacrimal de Schirmer (TLS), a avaliação microbiológica da conjuntiva ocular, a pressão intra-ocular (PIO) utilizando a tonometria de rebote e o comprimento horizontal da rima palpebral Neste estudo 84.1% das amostras analisadas foram positivas para crescimento microbiológico. Bactérias, fungos e hifas foram isolados e Staphylococcus spp. (21.9%) e Bacillus spp. (26.8%) foram isolados mais frequentemente. Os valores médios do teste de Lacrimal de Schirmer (TLS) foram de 7.6±4.6 e 6.6±4.4mm/min para olhos direito (OD) e esquerdo (OE), respectivamente (média = 7,11±0,76mm/min). A pressão intraocular média foi de 11.4±2.5 (OD) e 11.6±1.8mmHg (OE) anteriormente à anestesia (média 11,49±0,22 mmHg) e 7.6 ± 2.4 mmHg (OD) e 7.8 ± 1.8 mm Hg (OE) (média 7,71±0,08mm Hg) após a anestesia, verificando-se que a PIO foi significativamente menor quando os animais se encontravam sob anestesia comparativamente ao momento em que não estavam anestesiados. O comprimento horizontal da rima horizontal palpebral foi de 11.7±0.1mm OD e de 11.8±0.1mm OE (média 11,72±0,07mm). Verificou-se correlação positiva do TLS com o comprimento da fissura palpebral para a espécie estudada. Estes valores de referencia serão úteis no diagnóstico de alterações oculares da Arara Canindé.

Congenital disorder of glycosylation (CDG)-Ih patient with a severe hepato-intestinal phenotype and evolving central nervous system pathology.

We present the clinical, molecular, and biochemical diagnosis of a patient with congenital disorder of glycosylation (CDG)-Ih. We report significant brain dysfunction in this multisystem disease, further expanding its complex clinical spectrum.

Clinical and biochemical characterization of a patient with congenital disorder of glycosylation (CDG) IIx.

We describe a case of congenital disorder of glycosylation with chronic diarrhea, progressive liver cirrhosis, and recurrent infections. Transferrin analysis showed only hyposialylation, but analysis of total serum N-glycans indicated loss of additional sugars, arguing that the latter generates a more informative picture to search for the primary defect.

Clinical and molecular features of congenital disorder of glycosylation in patients with type 1 sialotransferrin pattern and diverse ethnic origins.

OBJECTIVE: To increase awareness of congenital disorders of glycosylation (CDG), we report the features of patients with a variety of clinical presentations ranging from mild hypotonia and strabismus to severe neurologic impairment. STUDY DESIGN: Nine North American patients with CDG type I and different ethnic origins were studied. RESULTS: All patients had transferrin isoelectric focusing studies with a type 1 sialotransferrin pattern. Molecular analysis showed the previously described R141H, V231M, and T237M PMM2 mutations in four patients as well as 3 rare mutations (DeltaC389, L104V, and IVS1 -1 G-->A) in the PMM2 gene in two Asian patients. CONCLUSIONS: The clinical features of these patients with diverse ethnic backgrounds confirm the variable course of CDG type I. Screening for CDG should be considered in children with relatively mild neurologic impairment, especially if they have suggestive findings such as cerebellar hypoplasia and abnormal fat distribution.

Two-way radio for rural health care delivery.

PIP: Over the past 6 years in Guyana, the MEDEX 2-way radio network has emerged as the most effective and reliable system of its kind. This 2-way radio system, originally designed to ease the communication difficulties of medical personnel in the field, now is fully incorporated into the administration of primary health care. The US Agency for International Development (AID) funded a pilot project from 1979-85 which established a dedicated 2-way radio system for the MEDEX program. The initial network of 10 sites has now been extended to 28 sites, with plans for at least 10 more. Site-to-site and site-to-headquarters distances range from 48 kilometers to in excess of 400 kilometers. The system design, chosen in consultation with the Guyana Telecommunications Corporation (GTC) is a low-powered system operating on 3 frequencies with sound quality similar to that of a taxi radio. During phase 1 of the project, portable generators were used at some sites and solar panels at others to provide the system's power. After a year it was found that fuel for the generators was expensive and difficult to obtain, and that some generators had broken down. No problems were encountered with the solar panels, so generator units were replaced with the more efficient solar installations. Each site is equipped with a fully transistorized 25-watt Stoner SSB-40A transceiver, a 3-element dipole antenna, a 12-volt automotive battery, a 5-watt solar panel for trickle-charging the battery, and a set of hand tools. To ensure that Guyanese personnel would be able to take full responsibility for the system, the project provided adequate training, local installation and maintenance, effective program management, and a 5-year stock of spare parts. A key element from the start has been involvement of GTC, whose technicians have been used to install and maintain the equipment under a contract with the Ministry of Health (MOH). A key person in the network is the full-time communications officer at MEDEX headquarters in Georgetown, who is resposible not only for communicating with the medex and controlling traffic on the network, but also for following up on their requests. This officer must locate a physician when an emergency call is received, determine the status of patients transferred to Georgetown or of delayed drug shipments, and provide other information upon request. The competence and dedication of this officer is vital to the successful operation of the network. The pattern of radio use varies depending upon regional needs. Between 1980-85, administrative uses of the network increased from 44 to 62% of all traffic, while medical calls declined in volume from 31 to 23%. 2-way radio has greatly improved rural primary health care delivery.^ieng