Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.     

Ontogeny, differentiation and growth of the endocrine pancreas

The pancreas develops from the primitive foregut endoderm, which differentiates into ductal, acinar and endocrine cells. This complex process is probably replicated in the adult pancreas when endocrine cell renewal is required, as may be the case in diabetes mellitus. This review describes what is known about the morphogenesis of the endocrine pancreas during ontogeny and the mechanisms regulating its differentiation and growth. Received: 23 December 1999 / Accepted: 15 February 2000     

Comparative typing of Campylobacter jejuni by heat-stable serotyping and PCR-based restriction fragment length polymorphism analysis

Campylobacter jejuni has become the most common bacterial cause of human gastroenteritis worldwide. Rapid, discriminatory typing methods are required to identify potential clusters of infections. The major disadvantage of the well-evaluated and widely used Penner heat-stable serotyping method is the high level of nontypeability. The correlation of the types determined by the Penner heat-stable serotyping method and PCR-based restriction fragment length polymorphism (RFLP) analysis of the lipooligosaccharide (LOS) biosynthesis genes of C. jejuni was studied with 149 C. jejuni strains. Of these strains, 79 were patient strains belonging to 25 Penner serotypes, 60 were nontypeable patient strains, and 10 were reference strains. A 9.6-kb DNA fragment of the LOS gene cluster was amplified and digested with the restriction enzymes HhaI and DdeI. Altogether, 39 different RFLP types (including 30 HhaI profiles and 32 DdeI profiles) were identified. Type Hh1Dd1 was the most common type, with 36% of the strains and strains of 12 serotypes being of this type. A high level of discrimination was obtained, and a correlation between the Penner serotypes and the PCR-RFLP types could be seen. Also, variation in the LOS biosynthesis genes within a single Penner serotype was found. Although the PCR-RFLP method may not be sufficient to compensate for Penner serotyping, it can give valuable information about nontypeable strains and further characterize strains of common serotypes.     

A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.     

A 10-Mb paracentric inversion of chromosome arm 2p inactivates MSH2 and is responsible for hereditary nonpolyposis colorectal cancer in a North-American kindred

Genomic deletions of the MSH2 gene are a frequent cause of hereditary nonpolyposis colorectal cancer (HNPCC), a common hereditary predisposition to the development of tumors in several organs including the gastrointestinal and urinary tracts and endometrium. The mutation spectrum at the MSH2 gene is extremely heterogeneous because it includes nonsense and missense point mutations, small insertions and deletions leading to frameshifts, and larger genomic deletions, the latter representing approximately 25% of the total mutation burden. Here, we report the identification and molecular characterization of the first paracentric inversion of the MSH2 locus known to cause HNPCC. Southern blot analysis and inverse PCR showed that the centromeric and telomeric breakpoints of the paracentric inversion map within intron 7 and to a contig 10 Mb 3' of MSH2, respectively. Pathogenicity of the paracentric inversion was demonstrated by conversion analysis. The patient's lymphocytes were employed to generate somatic cell hybrids to analyze the expression of the inverted MSH2 allele in an Msh2-deficient rodent cellular background. The inversion was shown to abolish MSH2 expression by both northern and western analysis. This study confirms that Southern blot analysis still represents a useful and informative tool to screen for and identify complex genomic rearrangements in HNPCC. Moreover, monoallelic expression analysis represents an attractive approach to demonstrate pathogenicity of unusual mutations in autosomal dominant hereditary conditions.     

Bi-allelic loss of function variants in SLC30A5 as cause of perinatal lethal cardiomyopathy

Perinatal mortality is a heavy burden for both affected parents and physicians. However, the underlying genetic causes have not been sufficiently investigated and most cases remain without diagnosis. This impedes appropriate counseling or therapy. We describe four affected children of two unrelated families with cardiomyopathy, hydrops fetalis, or cystic hygroma that all deceased perinatally. In the four patients, we found the following homozygous loss of function (LoF) variants in SLC30A5 NM_022902.4:c.832_836del p.(Ile278Phefs*33) and NM_022902.4:c.1981_1982del p.(His661Tyrfs*10). Knockout of SLC30A5 has previously been shown a cardiac phenotype in mouse models and no homozygous LoF variants in SLC30A5 are currently described in gnomAD. Taken together, we present SLC30A5 as a new gene for a severe and perinatally lethal form of cardiomyopathy.Subject terms: Cardiovascular diseases, Development, Medical genetics, Medical genomics     

Epidemiological and clinical features of Croatian children and adolescents with a PCR-confirmed coronavirus disease 2019: differences between the first and second epidemic wave

MethodsData on patients aged ≤19 years with a positive SARS-CoV-2 PCR test recorded in the period March 12-May 12 (first wave) and June 19-July 19, 2020 (second wave) were retrospectively analyzed. The periods were separated by several weeks with no incident cases.ResultsWe analyzed data on 289 children and adolescents (6.5% of all cases; incidence rate [IR] = 3.54, 95% confidence interval [CI] 3.14-3.97/million person-days), 124 in the first wave (IR = 2.27) and 165 in the second wave (IR = 6.37): IRR second/first = 2.71 (2.13-3.44). During the first wave, the incidence was highest in infants (IR = 3.48), while during the second wave it progressively increased to IR = 7.37 in 15-19-year olds. Family members were the key epidemiological contacts (72.6% cases), particularly during the first wave (95.8% vs 56.3%). Overall, 41.3% patients were asymptomatic, 25.3% in the first and 52.6% in the second wave. Age 15-19 years (vs younger) was associated with a higher (RR = 1.26, 1.02-1.54) and infection in the second wave with a lower probability (RR = 0.66, 0.53-0.81) of being symptomatic. The most common symptoms were fever, cough, and rhinorrhea. In children aged ≥7 years, headache, anosmia/ageusia, and sore throat were also recorded. Only one child suffered a severe disease. All but 18 (7.8%) children were treated only symptomatically, and all fully recovered.ConclusionA large proportion of SARS-CoV-2 PCR-positive children/adolescents were asymptomatic. The associated disease was predominantly mild, comparably so in the first and second pandemic wave.

Since the late December 2019, coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread quickly worldwide and as of early December accounts for more than 65 million cases diagnosed in more than 200 countries (1). At this point, the most affected countries in Europe are Russia, Spain, France, United Kingdom (UK), and Italy with consequently the highest mortality rates. The first case in Croatia was reported in the late February 2020, and within the next two months the infection expanded nationwide. During this first epidemic wave, Croatia was under a one-month lockdown, which rapidly decreased the disease incidence, and only a few newly diagnosed cases were reported between May 25 and June18, 2020. Easing of restrictions increased the incidence in late June, causing a second wave of COVID-19 in Croatia, with >147 000 cases reported so far (1,2).Over the last two decades, there were two other coronavirus outbreaks. Severe acute respiratory syndrome coronavirus appeared in 2002, affecting around 8000 people, with 10% mortality. Children (4 months-17 years) accounted for <0.02% of total cases, and there was no reported death in this age group. During the outbreak of the Middle East respiratory syndrome coronavirus, around 2300 people were infected, and children (<19 years of age) were rarely affected as well (2% of total cases; 2 reported deaths) (3,4). COVID-19 has exhibited a similar epidemiological pattern. Although early reports from China, Italy, and the United States (US) suggested that children and adolescents accounted for only 1%-2% of the overall COVID-19 cases (5-7), later reports around the world indicated a higher proportions of pediatric cases, between 1%-8% (8-10). Children of all ages can be affected by SARS-CoV-2 infection, but in contrast to other respiratory viruses, they usually suffer a mild or asymptomatic infection. Compared with adults, severe infections and fatal outcomes in children are rare, and several immunopathological mechanisms could be responsible for such differences in disease severity (11). Although many studies have reviewed the features of adults with COVID-19, overall data regarding pediatric cases are scarce, and most of them are reports from China and the US, with only a few studies describing disease in children from European countries.We aimed to describe epidemiological and clinical features of children and adolescents with COVID-19 confirmed by the polymerase chain reaction (PCR) test for SARS-CoV-2 in Croatia and to assess potential differences between the first (March-May 2020) and second (on-going) pandemic wave (June-July 2020).     

Induction of intervertebral disc-like cells from adult mesenchymal stem cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.