Next- generation sequencing is an effective method for diagnosing patients with different forms of monogenic diabetes.

AIM: Monogenic diabetes (MD) represents 5-7% of antibody-negative diabetes cases and is a heterogeneous group of disorders. METHODS: We used targeted next-generation sequencing (NGS) on Illumina NextSeq 550 platform involving the SureSelect assay to perform genetic and clinical characteristics of a study group of 684 individuals, including 542 patients referred from 12 Polish Diabetes Centers with suspected MD diagnosed between December 2016 and December 2019 and their 142 family members (FM). RESULTS: In 198 probands (36.5%) and 66 FM (46.5%) heterozygous causative variants were confirmed in 11 different MD-related genes, including 31 novel mutations, with the highest number in the GCK gene (206/264), 22/264 in the HNF1A gene and 8/264 in the KCNJ11 gene. Of the 183 probands with MODY1-5 diabetes, 48.6% of them were diagnosed at the pre-diabetes stage and most of them (68.7%) were on diet only at the time of genetic diagnosis, while 31.3% were additionally treated with oral hypoglycaemic drugs and/or insulin. CONCLUSIONS: In summary, the results obtained confirm the efficacy of targeted NGS method in the molecular diagnosis of patients with suspected MD and broaden the spectrum of new causal variants, while updating our knowledge of the clinical features of patients defined as having MD.

Three-dimensional high-resolution anorectal manometry: cut-off values for diagnosis of dyssynergic defecation in children.

OBJECTIVE: Dyssynergic defecation (DD) may be one of the most important causes of constipation, but its diagnostic criteria have not been formally validated in children. This study aims to evaluate constipated children with 3DHRAM (three-dimensional high-resolution anorectal manometry) and determine a new pediatric cut-off for DD variables. PATIENTS AND METHODS: 205 patients diagnosed with functional constipation (FC) based on Rome III criteria were prospectively enrolled. Data were compared to a historical control group (C). Initially, the diagnosis of DD was based on adult criteria and divided into 4 types. A new cut-off value for percent anal relaxation was determined based on ROC curve analysis. RESULTS: The FC group presented significantly lower values of percent anal relaxation during straining compared to the C group (9.5% vs. 20%, respectively, p=0.03). Based on adult criteria, DD was found in 53% of the FC group and 46% of the C group (p=0.3), with type II occurring most frequently (35.8%). New cut-off value of 31% for percent anal relaxation in children was derived based on the ROC curve analysis. Based on this new cut-off value, DD was diagnosed in 69.3% of constipated children, with type IV occurring most frequently (28.9%). The analysis of segmental pressure showed significant influence of segments at the locations of the puborectalis muscle and external anal sphincter. CONCLUSIONS: We found that during bear down maneuver the percent anal relaxation variable significantly differed between patients and controls. The higher cut-off value should be used when 3DHRAM and the standard four-type classification are used to diagnose DD in children.

Environmental factors driving the occurrence of the invasive cyanobacterium Sphaerospermopsis aphanizomenoides (Nostocales) in temperate lakes.

Cyanobacterial blooms are an increasing threat worldwide. Invasions of certain cyanobacterial species, mainly towards higher latitudes, add to this concern as they enrich the pool of potential bloom-formers in the invaded region. Among the numerous causes of this escalating process, climate warming is commonly considered the most crucial factor, but empirical studies of this issue are lacking. The aim of our study was to identify physical, chemical and biological factors related to the occurrence of an invasive cyanobacterium at the northern border of its putative current range, and thus enabling its expansion. This study focuses on the relatively little studied species Sphaerospermopsis aphanizomenoides (Nostocales, Cyanobacteria; synonyms: Aphanizomenon aphanizomenoides, Anabaena aphanizomenoides), which is predicted to become one of the main nuisance species of the future. Forty-nine freshwater lakes located between latitudes 51° and 55°N were examined for the presence of S. aphanizomenoides, and environmental factors that could drive its occurrence were studied simultaneously. To identify factors correlated with the presence of the species, principal component analysis (PCA) and Mann-Whitney U test were performed. Water temperature did not differentiate lakes with or without S. aphanizomenoides, however the study was conducted in a particularly hot summer. Total phosphorus concentration was identified as the primary driving factor of the occurrence of S. aphanizomenoides. The species grew in poor light conditions and high phytoplankton biomass, mainly in shallow lakes. As shown by detrended correspondence analysis (DCA), the species accompanied shade tolerant, eutrophic species of native and invasive cyanobacteria as well as eukaryotic algae. Our results indicate that eutrophication may be the primary factor enabling the increasing occurrence of S. aphanizomenoides in temperate environments, and suggest that this process may stimulate expansion of cyanobacterial species towards high latitudes.

Common polymorphism (81Val>Ile) and rare mutations (257Arg>Ser and 335Ile>Ser) of the MC3R gene in obese Polish children and adolescents.

The predisposing role to human obesity of the MC3R gene polymorphism is controversial. In this report we present the first study focused on the search for the MC3R polymorphism in the Polish population. Altogether 257 obese children and adolescents (RBMI>120) and 94 adults, who were never obese or overweight (BMI<25), were studied. For all subjects the entire coding sequence was analyzed by direct DNA sequencing. One common polymorphism (81Val>Ile) and two rare mutations (257Arg>Ser and 335Ile>Ser) were identified. The common polymorphism was widely distributed in the obese and control cohorts, while the mutations were identified in four obese subjects only. In case of the 335Ile>Ser substitution a three-generation family, consisting of 20 members, was also analyzed. It was found that all carriers of the 335Ser mutation were obese, but among non-carriers obese subjects also were found. Our study suggests that the predisposing effect to obesity of the 81Ile polymorphic variant is rather unlikely. With regard to the studied rare mutations we suggest that the 335Ser allele may have a small predisposing effect.

Effect of three common SNPs in 5'-flanking region of LEP and ADIPOQ genes on their expression in Polish obese children and adolescents.

Genes encoding adipokines are considered as candidates for human obesity. In this study we analyzed the expression of leptin (LEP) and adiponectin (ADIPOQ) genes in relation to common 5'-flanking or 5'UTR variants: -2548G>A (LEP), 19A>G (LEP) and -11377C>G (ADIPOQ) in Polish obese children and adolescents. Relative transcription levels in the subcutaneous adipose tissue (real time RT-PCR) and serum protein concentrations (RIA) were measured in 48 obese subjects with known genotypes at three polymorphic sites and in five non-obese controls. None of the studied polymorphisms altered significantly the expression. Significantly elevated relative transcription levels of the LEP gene (P < 0.05) and serum leptin concentrations (P < 0.01) were recorded in obese patients, when compared with the non-obese controls, but such differences were not found for the ADIPOQ gene. Interestingly, the leptin to adiponectin protein concentration ratio (L/A) was approximately sevenfold higher in obese children and adolescents when compared with the non-obese controls (P < 0.001). Taking into consideration the observed relationship between the genotypes and the gene expression level we suggest that these SNPs are not conclusive markers for predisposition to obesity in Polish children and adolescents. On the other hand, we confirmed that the leptin to adiponectin gene expression ratio (L/A) is an informative index characterizing obesity.

First and second line mechanisms of cadmium detoxification in the lichen photobiont Trebouxia impressa (Chlorophyta).

"First line" defence mechanisms, such as phytochelatin biosynthesis, and "second line" mechanisms, such as stress protein induction, were investigated in cadmium-exposed cells of Trebouxia impressa Ahmadjian, a green microalgal species that is a common photobiont of the lichen Physcia adscendens (Fr.) H. Olivier. When T. impressa cells were exposed to 0, 9 and 18 microM Cd for 6, 18 and 48 h, glutathione and phytochelatins efficiently protected the cells against Cd damage. By contrast, the highest Cd concentration (36 microM) at the longest exposure-time (48 h) caused marked drops in glutathione and phytochelatin content, several types of ultrastructural damage, and decreases in cell density and total chlorophyll concentration. In this case, induction of stress proteins was observed, but only long after the induction of phytochelatins. Thus, stress proteins could represent a "second line" mechanism to counteract Cd stress, activated when there is a decline in the "first line" mechanism of Cd detoxification given by phytochelatins.

Arsenic availability, toxicity and direct role of GSH and phytochelatins in As detoxification in the green alga Stichococcus bacillaris.

Accumulation and toxicity of inorganic arsenic forms As(III) and As(V) to the green microalga Stichococcus bacillaris depended on environmental variables. pH of exposure and to a lesser extent elevated concentrations of humic acid, chloride and orthophosphate ions affected arsenic accumulation and its toxicity. As(V) was more toxic than As(III), especially at the near neutral pH 6.8. Intracellular As(V) uptake by algal cells was greater at pH 6.8 than at 8.2. In response to As(III) and As(V) the alga produced phytochelatins (PC(2-3)), but at As(V) exposure, their levels in cells were higher than with As(III), suggesting higher As(V) than As(III) availability and uptake. Arsenic in algal cells occurred in various complexes with non-protein SH groups. Some of these complexes dissociated under acidic conditions, but others were able to dissociate only at an alkaline pH. The former consisted of SH groups of phytochelatins. Those dissociating at an alkaline pH involved SH groups from both glutathione (GSH) and phytochelatins (PC) or their derivatives. In the predominant acid-stable mixed As-SH complex, the ratio of SH (PC(2)) to SH (GSH) was 2:1, which suggests that one molecule of PC(2) (containing two SH groups) together with one molecule of GSH were involved in intracellular complexation of each As atom. This is the first demonstration of GSH involvement in arsenic complexation, in vivo. The intracellular concentration of As was greater than that of non-protein SH groups which suggests that not all the arsenic in algal cells was complexed and detoxified by thiol groups.

Phytochelatin production in freshwater algae Stigeoclonium in response to heavy metals contained in mining water; effects of some environmental factors.

Production of phytochelatins (PC) in two freshwater, filamentous green algae of the genus Stigeoclonium, in response to heavy metals contained in mining water was studied. Stigeoclonium sp. grown abundantly in ditches with the mining water (southern Poland) accumulated high amounts of heavy metals. The other studied alga Stigeoclonium tenue Kütz. was isolated from unpolluted lake water in the Netherlands. Both algae exposed to the heavy metal mixture (17 microM; mainly zinc) contained in the hard, alkaline (pH 8.2) mining water produced similar amounts of phytochelatins (PC2 and PC3): 500-600 nmol SH g(-1) dry weight. After water acidification to pH 6.8, a > 2-fold increase of the total phytochelatin level as well as the appearance of longer chain peptide PC4 in the cells of both algae was observed. The concentration of labile forms of zinc in the acidified mining water was four times as high as that in the alkaline water. The heavy metal mixture (17 microM) in non-complexing solution of pH 6.8 caused a comparable phytochelatin production in both Stigeoclonium strains as the same mixture present in the acidified mining water. However, in the non-complexing solution of pH 8.2, the metal mixture induced in algae more PCs than at pH 6.8. A positive effect of bicarbonate enrichment in the solution on the PC production in S. tenue was also observed. Stigeoclonium sp. exposed to high concentrations (10 microM) of individual metals (Zn, Pb and Cd available as free cations) synthesised much higher amounts of phytochelatins (PC2-PC4) than in response to the metal mixture contained in the mining water. The order of PC induction by the studied metals in the Stigeoclonium sp. was Cd > Pb > Zn. Addition of suspended solid matter to the heavy metal solution resulted in essential quantitative changes in phytochelatins in algal cells; a considerable decrease of PC2, PC3 and PC4 levels was observed. The PC production in algae of the genus Stigeoclonium exposed to the heavy metal mixture is discussed in the context of various metal bioavailability and the algal adaptation to complex aquatic environment.

The impact of inorganic tin on the planktonic cyanobacterium Synechocystis aquatilis: the effect of pH and humic acid.

The influence of inorganic tin compounds on the unicellular cyanobacterium Synechocystis aquatilis was studied, and its dependence on changing pH of the surrounding medium and the presence of humic acid. Both Sn(II) and Sn(IV), used as chlorides (at the concentrations 1-10 mg litre(-1)), inhibit the growth and chlorophyll a content of the cyanobacterium cultures, but only under alkaline conditions. Generally, the observed tin toxicity increased with increase of metal concentration, time of exposure and pH value of the medium (in the range 7-9.8). Sn(II) seems to be more toxic than Sn(IV). At the lowest studied metal concentration (1 mg litre(-1)), Sn(II) caused a 36 and 40% decrease in growth and chl a content, respectively, after 96 h exposure at pH 9.8, while Sn(IV) caused even a slight increase of both physiological parameters (hormetic effect). Similar increases in growth and chl a content were also observed at a high Sn (II) and Sn(IV) concentration (10 mg litre(-1)), but only in cultures exposed to metal at pH 7. At high pH (9.8), 10 mg litre(-1) of Sn(II) and Sn(IV) significantly suppressed both the growth of the cyanobacterium (by 54.2 and 26.1%, respectively) and the chl a content in cultures (by 58.2 and 24%, respectively). Humic acid reduced the toxicity of tin towards the cyanobacterium. The observed effects of pH and complexing ligand on the inorganic tin toxicity are discussed in the context of changing, chemical metal speciation and bioavailability.