Clinical Case Report of Non-Diabetic Hypoglycemia Due to a Combination of Germline Mutations in the MEN1 and ABCC8 Genes.

INTRODUCTION: Non-diabetic hypoglycemia (NDH) is a collective term including the multiple causes of hypoglycemic syndrome not due to diabetes mellitus. NDH may result from insulinoma, IGF-2-omas, hypocorticism, Hirata's disease, genital disorders of glucose metabolism, etc. One of the most common causes of NDH faced by an endocrinologist is insulinoma, which in turn can be part of the hereditary syndrome of multiple endocrine neoplasia type 1 (MEN1). Congenital disorders of glucose metabolism in adult patients, on the contrary, are diagnosed extremely rarely, since they usually manifest in childhood. This article presents a unique clinical case of a patient with NDH and genetically verified MEN1 in combination with congenital hyperinsulinism due to an ABCC8 gene mutation. CASE REPORT: A 43-year-old patient with hypoglycemic symptoms from childhood is presented, in whom multiple pancreatic tumors and fluctuations in glycemia from 38.7 mg/dL to 329.7 mg/dL (2.15 to 18.3 mmol/L) were detected in adulthood, but a mild course of hypoglycemic syndrome was noted. Numerous examinations that were performed to establish an accurate diagnosis are described, signs that served as a reason for expanding the complex of studies are indicated, possible pathogenetic mechanisms of the mild course of hypoglycemic syndrome and hyperglycemic conditions are discussed. CONCLUSION: This case report is original and highlights that we must always remain intolerant of the inexplicable. Conducting an extended gene study can help perform a correct diagnosis in complex cases.

Quality Assessment of Processed Graphene Chips for Biosensor Application.

The quality of graphene intended for use in biosensors was assessed on manufactured chips using a set of methods including atomic force microscopy (AFM), Raman spectroscopy, and low-frequency noise investigation. It is shown that local areas of residues on the graphene surface, formed as a result of the interaction of graphene with a photoresist at the initial stage of chip development, led to a spread of chip resistance (R) in the range of 1-10 kOhm and to an increase in the root mean square (RMS) roughness up to 10 times, which can significantly worsen the reproducibility of the parameters of graphene chips for biosensor applications. It was observed that the control of the photoresist residues after photolithography (PLG) using AFM and subsequent additional cleaning reduced the spread of R values in chips to 1-1.6 kOhm and obtained an RMS roughness similar to the roughness in the graphene film before PLG. Monitoring of the spectral density of low-frequency voltage fluctuation (SU), which provides integral information about the system of defects and quality of the material, makes it possible to identify chips with low graphene quality and with inhomogeneously distributed areas of compressive stresses by the type of frequency dependence SU(f).

Serum circulating miRNA-342-3p as a potential diagnostic biomarker in parathyroid carcinomas: A pilot study.

OBJECTIVE: To compare the serum miRNA expression profiles between patients with benign and malignant parathyroid tumours. BACKGROUND: Despite recent advances in molecular biology, a histological tissue biopsy is still the method of choice used to diagnose most cancers. The preoperative cytology is not an applicable method for diagnosis of parathyroid cancer (PC); therefore, huge interest exists in terms of finding alternative methodologies to seek specific cancer biomarkers. DESIGN: A retrospective cross-sectional study. PATIENTS AND METHODS: Serum samples of patients with PC (n = 13) and parathyroid adenoma (PA) (n = 11), age (p = .999) and sex (p = .999) were matched and examined via the simultaneous comparative expression analysis of 754 microRNAs (miRNAs). The «TaqMan OpenArray Human MicroRNA Panel¼ (Applied Biosystems) was used to conduct real-time PCRs using the «QuantStudio 12К Flex¼ station (Life Technologies). RESULTS: According to the results of a pilot study, significant changes in expression levels between the PC group and the PA group (control) (p < .05) were observed for 17 miRNAs. Among them, the downregulation of miRNA-342-3p met the Benjamini-Hochberg adjustment criteria for multiple comparisons (p = .02). CONCLUSIONS: Serum miRNA-342-3p could be a promising biomarker for PC to improve diagnosis and prognosis.

Successful Use of Denosumab for Life-Threatening Hypercalcemia in a Pediatric Patient with Primary Hyperparathyroidism.

INTRODUCTION: Primary hyperparathyroidism (PHPT) is rare and usually symptomatic in children. There is no approved medication to lower serum calcium levels in this patient group. Denosumab is used in adult patients with osteoporosis and hyperparathyroidism. To our knowledge, only 1 case of denosumab treatment in a child with severe PHPT has been reported to date. CASE PRESENTATION: A 16-year-old female was referred to our clinic with symptoms including pathologic fractures, nausea, emesis, and progressive weight loss. At admission, her serum total calcium was 4.17 mmol/L (reference range 2.15-2.55), parathyroid hormone 2,151 pg/mL (15-65), and phosphate 1.07 mmol/L (1.45-1.78). Due to potentially life-threatening hypercalcemia, denosumab 60 mg subcutaneously was administered after obtaining informed consent. Serum calcium levels were reduced within 12 h of injection and the patient's condition rapidly improved, which allowed genetic testing to be done prior to surgery. A heterozygous mutation in the CDC73 gene was revealed, and a parathyroidectomy was performed on day 22 after denosumab administration. Morphological examination revealed solitary parathyroid adenoma. After surgery, hypocalcemia developed requiring high doses of alfacalcidol and calcium supplements. CONCLUSION: Our case supports the previous observations in adults that denosumab can be safely and effectively used as a preoperative treatment in patients with PHPT and severe hypercalcemia and shows that it may be used in pediatric patients.

Genomes of the T4-related bacteriophages as windows on microbial genome evolution.

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

Genetic insertions and diversification of the PolB-type DNA polymerase (gp43) of T4-related phages.

In Escherichia coli phage T4 and many of its phylogenetic relatives, gene 43 consists of a single cistron that encodes a PolB family (PolB-type) DNA polymerase. We describe the divergence of this phage gene and its protein product (gp43) (gene product 43) among 26 phylogenetic relatives of T4 and discuss our observations in the context of diversity among the widely distributed PolB enzymes in nature. In two T4 relatives that grow in Aeromonas salmonicida phages 44RR and 25, gene 43 is fragmented by different combinations of three distinct types of DNA insertion elements: (a) a short intercistronic untranslated sequence (IC-UTS) that splits the polymerase gene into two cistrons, 43A and 43B, corresponding to N-terminal (gp43A) and C-terminal (gp43B) protein products; (b) a freestanding homing endonuclease gene (HEG) inserted between the IC-UTS and the 43B cistron; and (c) a group I intron in the 43B cistron. Phage 25 has all three elements, whereas phage 44RR has only the IC-UTS. We present evidence that (a) the split gene of phage 44RR encodes a split DNA polymerase consisting of a complex between gp43A and gp43B subunits; (b) the putative HEG encodes a double-stranded DNA endonuclease that specifically cleaves intron-free homologues of the intron-bearing 43B site; and (c) the group I intron is a self-splicing RNA. Our results suggest that some freestanding HEGs can mediate the homing of introns that do not encode their own homing enzymes. The results also suggest that different insertion elements can converge on a polB gene and evolve into a single integrated system for lateral transfer of polB genetic material. We discuss the possible pathways for the importation of such insertion elements into the genomes of T4-related phages.

Plasticity of the gene functions for DNA replication in the T4-like phages.

We have completely sequenced and annotated the genomes of several relatives of the bacteriophage T4, including three coliphages (RB43, RB49 and RB69), three Aeromonas salmonicida phages (44RR2.8t, 25 and 31) and one Aeromonas hydrophila phage (Aeh1). In addition, we have partially sequenced and annotated the T4-like genomes of coliphage RB16 (a close relative of RB43), A. salmonicida phage 65, Acinetobacter johnsonii phage 133 and Vibrio natriegens phage nt-1. Each of these phage genomes exhibited a unique sequence that distinguished it from its relatives, although there were examples of genomes that are very similar to each other. As a group the phages compared here diverge from one another by several criteria, including (a) host range, (b) genome size in the range between approximately 160 kb and approximately 250 kb, (c) content and genetic organization of their T4-like genes for DNA metabolism, (d) mutational drift of the predicted T4-like gene products and their regulatory sites and (e) content of open-reading frames that have no counterparts in T4 or other known organisms (novel ORFs). We have observed a number of DNA rearrangements of the T4 genome type, some exhibiting proximity to putative homing endonuclease genes. Also, we cite and discuss examples of sequence divergence in the predicted sites for protein-protein and protein-nucleic acid interactions of homologues of the T4 DNA replication proteins, with emphasis on the diversity in sequence, molecular form and regulation of the phage-encoded DNA polymerase, gp43. Five of the sequenced phage genomes are predicted to encode split forms of this polymerase. Our studies suggest that the modular construction and plasticity of the T4 genome type and several of its replication proteins may offer resilience to mutation, including DNA rearrangements, and facilitate the adaptation of T4-like phages to different bacterial hosts in nature.

Genetic diversity among five T4-like bacteriophages.

BACKGROUND: Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank. RESULTS: Here, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR) and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved genes common to these genomes as well as hundreds of additional open reading frames (ORFs) that are nonconserved. Although some of these ORFs resemble known genes from bacterial hosts or other phages, most show no significant similarity to any known sequence in the databases. The five genomes analyzed here all have similarities in gene regulation to T4. Sequence motifs resembling T4 early and late consensus promoters were observed in all five genomes. In contrast, only two of these genomes, RB69 and 44RR, showed similarities to T4 middle-mode promoter sequences and to the T4 motA gene product required for their recognition. In addition, we observed that each phage differed in the number and assortment of putative genes encoding host-like metabolic enzymes, tRNA species, and homing endonucleases. CONCLUSION: Our observations suggest that evolution of the T4-like phages has drawn on a highly diverged pool of genes in the microbial world. The T4-like phages harbour a wealth of genetic material that has not been identified previously. The mechanisms by which these genes may have arisen may differ from those previously proposed for the evolution of other bacteriophage genomes.

Divergence of the mRNA targets for the Ssb proteins of bacteriophages T4 and RB69.

The single-strand binding (Ssb) protein of phage T4 (T4 gp32, product of gene 32) is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor. We also show that the natural mRNA targets (translational operators) for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32. Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors. These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor.

Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins.

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant (Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-). Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes revealed the patterns and efficiencies of proofreading, while Tyr(567) was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.

RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69.

The DNA polymerases (gp43s) of the two related phages T4 and RB69 are DNA-binding proteins that also function as mRNA-binding autogenous translational repressors. As repressors, T4 gp43 is narrowly specific to its own mRNA whereas RB69 gp43 is equally effective against mRNA for either protein. We used in vitro RNase-sensitivity and RNA footprinting assays to identify features of the non-identical T4 and RB69 mRNA targets (translational operators) that allow for their identical binding affinities and biological responses to RB69 gp43. We observed that T4 gp43 and RB69 gp43 produce identical footprints on RNA substrates bearing the T4-derived operator, suggesting that the two gp43s make identical contacts with this operator. In contrast, the footprint produced by RB69 gp43 on its autogenous RNA target was shorter than its footprint on operator RNA from T4. As expected, we also observed only weak protection of RB69-derived operator RNA from RNase by T4 gp43; however, photocross-linking studies suggested that T4 gp43 recognizes structural features of the RB69-derived operator that are not detected by RNase- sensitivity assays. The results suggest that RB69 gp43 and T4 gp43 differ in their abilities to use RNA-sequence-independent interactions to configure potential RNA targets for translational repression.

Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69.

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.