Microbial communities as indicators of marine ecosystem health: Insights from coastal sediments in the eastern Adriatic Sea.

Considering the adaptability and responsiveness of microorganisms to environmental changes, their indicator potential is still not acknowledged in European directives. This comprehensive study examined the changes of microbial communities in sediments and a range of geochemical parameters from pristine and anthropogenically impacted coastal areas in the eastern Adriatic Sea. Various analytical methods found evidence of sediment contamination (high toxicity level, enrichments of metals, tributyltin) in certain areas, leading to the categorization of sediments based on the level of anthropogenic disturbance. Prokaryotes were identified as the most promising group of microbes for further research, with specific bacterial families (Rhodobacteraceae, Ectothiorhodospiraceae, Cyclobacteriaceae) and genera (Boseongicola, B2M28, Subgroup 23, Sva0485, Thiogranum) proposed as potential indicators of environmental status. Finally, predictive models were developed to identify key indicator variables for assessing anthropogenic impact in sediments. This research represents an essential step toward incorporating microbial communities into assessments of benthic environmental health.

Prevalence of HEV RNA in Croatian blood donors.

OBJECTIVES: HEV infection is asymptomatic for immunocompetent blood donors (BD). Transfused HEV-infected blood products may cause potentially hazardous HEV infection in immunocompromised patients. Evaluation of the need for routine BD HEV RNA screening primarily demands the establishment of HEV infection prevalence in Croatian BD. MATERIALS AND METHODS: We tested BD samples in ID-NAT with the Procleix UltrioPlex E screening test for simultaneous detection of HBV DNA, HCV RNA, HIV-1,2 RNA, and HEV RNA (Grifols, Spain). HEV infection was confirmed with HEV RNA quantitative test (Altona Diagnostics, Germany) and HEV IgM and HEV IgG antibody test (DIA.PRO Diagnostic Bioprobes, Italy). We analysed the HEV RNA sequence and performed a phylogenetic analysis. We recorded BD's anamnestic data and dietary habits. BDs gave follow-up samples after two months and did not donate blood for six months. RESULTS: Between December 2021 and March 2022, we tested 8,631 donations and found four HEV RNA-positive donations, which equals to one in 2,158 donations (0.046 %, 95 % confidence interval, 0.018 %-0.119 %). Confirmatory HEV RNA testing gave results from negative to 4.73E + 3 IU/ml HEV RNA. Three donations were in the serological window period. We have genotyped HEV RNA of two infected BD as genotype HEV-3c. Blood donors didn't report any health problems and their diet included pork. Testing on follow-up samples presented seroconversion and no HEV RNA could be detected. CONCLUSION: The incidence of HEV RNA infection in BD in Croatia corresponds with other European data. The decision on implementation of HEV NAT screening in Croatia needs an expert team evaluation of the possible risk of TT-HEV infection.

Human neutrophil antigen-1, -3, -4, and -5 allele and genotype frequencies in the Croatian blood donor population and their clinical significance.

OBJECTIVES: Human neutrophil antigens (HNAs) and antibodies play an important role in allo- and autoimmunity associated with immune neutropenia and transfusion reactions. The aim of this study was to determine the HNA-1, -3, -4 and -5 allele and genotype frequencies in the Croatian blood donor population to assess the role of HNA-1, -3, -4, and -5 alleles in the development of neonatal alloimmune neutropenia and antibody-mediated transfusion-related acute lung injury. MATERIAL AND METHODS: A total of 371 blood samples from unselected healthy blood donors were analyzed. Samples from all 371 donors were genotyped for HNA-1, samples from 160 donors were genotyped for HNA-3, and samples from 142 donors were genotyped for HNA-4 and HNA-5 using the polymerase chain reaction with sequence-specific primers (PCR-SSP) method. RESULTS: The frequencies of the FCGR3B*01, FCGR3B*02 and FCGR3B*03 HNA-1 alleles were 0.393, 0.607 and 0.022, and of the SLC44A2*01 and SLC44A2*02 HNA-3 alleles 0.781 and 0.219, respectively. The frequencies of the ITGAM*01 and ITGAM*02 HNA-4 alleles were 0.796 and 0.204, and of the ITGAL*01 and ITGAL*02 HNA-5 alleles 0.718 and 0.282, respectively. CONCLUSION: These are the first results on the HNA allele and genotype frequencies in the Croatian blood donor population. We observed no deviations from previous reports on Caucasian populations. Determination of the HNA antigen frequencies in the population is important to estimate the risk of alloimmunization to HNA, especially the risk of fetal-maternal incompatibility and alloantibody production by transfusion of the HNA incompatible blood components.

Transient pseudothrombocytopenia (PTCP) in the neonate due to the mother.

Thrombocytopenia with platelet count <50×109/L is common laboratory finding in a severely ill newborn in neonatal intensive care units (ICU). Neonates with severe thrombocytopenia are at risk of bleeding. Most dangerous is intracerebral hemorrhage (ICH) frequently leading to death or lifelong neurological sequels. Pseudothrombocytopenia (PTCP) is a rare in vitro phenomenon of falsely low platelet count determined on hematology analyzers due to platelet clumping in ethylenediaminetetraacetic acid (EDTA) anticoagulated blood. PTCP was also reported in pregnant women with isolated thrombocytopenia. EDTA-PTCP in the neonate due to the transplacental transmission of maternal antibodies has been reported only in a few cases. Although PTCP is rare phenomenon, it should always be excluded in newborns with isolated thrombocytopenia to avoid erroneous interpretation of platelet and leukocyte count, unnecessary laboratory investigation of false positive antiplatelet antibodies and needless platelet transfusions. We report on two cases of transient PTCP in a neonate due to transplacental transfer of maternal EDTA-dependent autoantibodies of IgG class from the same mother.

Prenatal RHD genotyping in Croatia: preliminary results.

OBJECTIVES: Croatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed. MATERIALS AND METHODS: Evaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12-36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and ß-actin genes digested with BsTUI were used. RESULTS: We identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively. CONCLUSION: Automated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.

The importance of combined 24-hour multichannel intraluminal impedance-pH monitoring in the evaluation of children with suspected laryngopharyngeal reflux.

OBJECTIVE: The aim of this study was to investigate the diagnostic usefulness of combined multichannel intraluminal impedance-pH (MII-pH) monitoring in children with suspected laryngopharyngeal reflux (LPR). DESIGN, SETTING AND PARTICIPANTS: A prospective study including children in whom, due to LPR suggestive symptoms, MII-pH monitoring was performed at tertiary medical centre from February 2012 to July 2015. INTERVENTIONS: All included children underwent same diagnostic protocol which included examination by single pulmonologist and ENT specialist and underwent 24-hour MII-pH monitoring. MAIN OUTCOMES: Primary outcome was to determine MII-pH characteristics of the children in whom LPR was suspected based on symptoms and ENT examination. RESULTS: One hundred and four patients (mean age 8.9 years; range 0.4-17.9 years; male/female 57/47) participated in the study. In children with signs and symptoms suggestive of LPR, MII-pH monitoring found the median incidence of proximal gastro-oesophageal reflux (GER) of 15 (range 0-129), proximal acidic GER of 6.5 (range 0-66) and weakly acidic GER of 5 (range 0-102). There were significant positive correlations between the number of GER (proximal total, acidic and weakly acid) with Reflux Finding Score, Reflux Symptom Index and presence of eosinophils in nasal swabs. The only endoscopy ENT finding which significantly correlated with total proximal GER, acid proximal GER and weakly acidic proximal GER was arytenoid hyperaemia. CONCLUSION: Both acid and non-acid reflux seem to have a significant role in the pathogenesis of LPR.

Intraplacental villous artery resistance indices and identification of placenta-mediated diseases.

OBJECTIVE: Placenta-mediated diseases (PMDs) including preeclampsia and fetal growth restriction are often characterized by shallow trophoblast invasion and incomplete spiral artery remodeling leading to impaired placental perfusion. In this context, umbilical artery (UA) Doppler can be used to detect high resistance to flow characteristic of very late-stage placental disease. We propose that evaluation of intraplacental villous artery (IPVA) resistance can provide earlier detection of increased resistance in placental flow. STUDY DESIGN: Seventy-five patients were recruited from the Ottawa Hospital. All had scans at 18 to 20, 28 and 34 weeks of gestation. IPVAs arising perpendicular to the chorionic plate in three regions (placental tips 4 cm away from cord insertion and within 1 cm from cord insertion) were sampled at each gestational age for resistance index (RI) and pulsatility index (PI). UA Doppler was also obtained from a free loop of cord. Pregnancy outcomes were collected from a chart review. Data were analyzed using SAS version 9.4 and standard statistic tests (mean±s.d., Student's t-test, mixed-effects modeling). RESULT: A total of 53 patients completed the study. Of these, 38 had normal pregnancy outcomes (controls) and 15 (cases) developed PMD (preeclampsia, n=8 and low birth weight/intrauterine growth restriction, n=7). Mean birth weight in the study group was 2482.1±518.85 g. At 18 to 20, 28 and 34 weeks gestation, the mean IPVA resistance indices in the control group were 0.86±0.16, 0.81±0.12 and 0.71±0.12 for PI and 0.57±0.07, 0.55±0.06 and 0.49±0.06 for RI, respectively. However, in the cases developing PMDs, the PIs were 1.09±0.17, 0.95±0.21 and 0.78±0.07 and RIs 0.66±0.07, 0.60±0.07 and 0.54±0.04, respectively (P<0.05). UA PI and RI Doppler did not differ between the groups as early as 18 to 20 weeks gestation. CONCLUSION: Doppler measures of IPVA appear superior to UA in detecting early changes related to PMD. IPVA PI and RI Doppler may be useful in the early identification of patients at risk of PMD.

Prediction of difficulties in laparoscopic cholecystectomy on the base of routinely available parameters in a smaller regional hospital.

BACKGROUND: Identification of preoperative factors that indicate difficulties in the operation are in the function of primary prevention of intraoperative complications and require selection of an experienced surgical team, planning of operating program and timely provision of information to patients about the increased likelihood of conversion. AIM: Identification of preoperative factors of operative difficulties by analysis of routine clinical parameters. PATIENTS AND METHODS: A prospective cohort study of patients who underwent laparoscopic cholecystectomy from February 2005 to December 2009. All patients were operated by the same surgeon. There were 369 operated patients. Conversion was done in 10 patients. Main outcome measures were: duration of stages of laparoscopic cholecystectomy and conversion; identification of predictive parameters; assessment of their predictive value; assessment of the predictive value of individual parameters in respect to the duration of stages of laparoscopic cholecystectomy; correlation of parameter predictive value and duration of laparoscopic cholecystectomy stage. RESULTS: Multivariate stepwise linear regression analysis showed that previous history of acute cholecystitis, gallbladder wall thickness ˃ 4 mm, acute cholecystitis to admission, size of calculus > 2 cm, > 5 attacks of pain that lasted longer of 4 hours, diabetes mellitus, duration of symptoms longer then 36 months and pericholecystic fluid collection were significant for prediction of difficulties of laparoscopic cholecystectomy. CONCLUSIONS: Based on clinical, laboratory and ultrasonographic parameters without the use of highly sophisticated diagnostic procedures and increasing medical costs it is possible to predict difficulties in the laparoscopic cholecystectomy.

Substantial bone loss in the mandibular central incisors area as a complication of tongue piercing: a case report.

BACKGROUND: Along with the increasing popularity of oral piercings, the number of reported complications and side effects increases, too. CASE REPORT: The aim of this report is to present a case of substantial bone loss in the area of the mandibular central incisors caused by lingual piercing and persistent bad habits. Dentist should be aware of potential complications associated with oral piercings and warn patients about them.

Identification and characterization of potential autochthonous starter cultures from a Croatian "brand" product "Slavonski kulen".

The microbial population of a traditional Croatian fermented sausage "Slavonski kulen" was isolated, identified and subjected to technological and functional characterization in order to select potential autochthonous functional starter cultures. Dominant microflora were lactic acid bacteria (LAB), followed by staphylococci. Identification of isolated lactobacilli showed domination of Leuconostoc mesenteroides and Lactobacillus acidophilus while Staphylococcus xylosus and Staphylococcus warneri outnumbered the staphylococcal microbiota. Most of the isolated LAB and Staphylococcus species displayed good growth in the presence of 5% of NaCl and at 12, 18 and 22°C. All LAB and most of the staphylococci possess proteolytic activity and only Staphylococcus xylosus had lipolytic activity. All lactobacilli and staphylococci isolates produced significant concentrations of lactic acid (as determined by HPLC) and showed antimicrobial activity against pathogenic test microorganisms. Dominant LAB and Staphylococcus species displayed growth in the presence of 1% bile. Most of the staphylococci and all of lactobacilli showed sensitivity to all antibiotics tested.

Severe psychomotor retardation in a boy with a small supernumerary marker chromosome 19p.

We describe the clinical case of a nine-year-old boy with psychomotor retardation and a small supernumerary marker chromosome (sSMC) present in mosaic form. Fluorescence in situ hybridization (FISH) using centromere cross-hybridizing probes D1/5/19Z (pZ5.1), the whole chromosome paint probe 19, pool YACs19p (839B1, 872G3, 728C8), and pool YACs19q (767C4, 761C1, 786G6) demonstrated that the sSMC was derived from chromosome 19p. Based on GTG-banding and FISH analyses, the patient's karyotype was interpreted as: 47,XY,+mar.ish der(19) (:p13.3-->p11:)(839B1+, 872G3+,728C8+, D1/5/19Z+) de novo[52]/46,XY[48]. To our knowledge, only two other similar cases have been reported. This case helps to better delineate karyotype-phenotype correlations between sSMC 19p and associated clinical phenomena.

SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis.

Sam68 (Src associated in mitosis; 68 kDa) is an RNA-binding protein and substrate of Src family kinases. It is thought to play a role in cell cycle progression. Overexpression of Sam68 in fibroblasts was reported to have two separable functions dependent on its ability to bind RNA--cell cycle arrest or the induction of apoptosis. Post-translational modification with SUMO (small ubiquitin-like modifier) is common to many transcription factors and can regulate protein localization, stability and function. Here we show Sam68 to be modified by SUMO, and demonstrate that the SUMO E3 ligase (PIAS1) (protein inhibitor of activated STAT1) can enhance Sam68 sumoylation. Lysine 96, the first lysine in the amino-terminal region of Sam68, was found to be the major SUMO acceptor site. Mutation of the SUMO acceptor lysine to arginine enhanced the ability of Sam68 to induce apoptosis but inhibited its ability to act as a transcriptional inhibitor of cyclin D1 expression. A SUMO-1 Sam68 fusion protein, on the other hand, inhibited the ability of Sam68 to induce apoptosis but was a strong repressor of cyclin D1 expression. Thus, SUMO may be an important regulator of Sam68 function in cell cycle progression.

Clinical validation of bronchial hyperresponsiveness, allergy tests and lung function in the diagnosis of asthma in persons with dyspnea.

The aim of the study was to determine the most useful tests for decision making in the diagnosis of asthma in patients with dyspnea assessed by commonly used terms: sensitivity, specificity, positive and negative predictive values and diagnostic accuracy. In a group of 195 patients with dyspnea data were analyzed with respect to case histories and different diagnostic procedures: bronchial hyperresponsiveness (BHR), skin prick tests (SPT), total IgE, spirometry (FEV1), sputum eosinophils (SE) and blood eosinophilia (BE). Asthma was diagnosed in 141 subjects. The control group comprised 18 subjects. Sensitivity for BHR in asthma in subjects with dyspnea amounted to 97%, for SPT to 62%, while all other diagnostics were lower than 50%. Specificity was highest for SE (94%), and BHR (85%). Positive predictive value (PPV) in asthma was for BHR 94%, for SE 86%, for SPT 81%, for decreased FEV1 79%, total IgE 72% and BE 64%. The highest negative predictive value (NPV) was found for BHR (92%). Diagnostic accuracy was highest for BHR 93% and for SPT 62%, while all other tests were comparable or lower than 50%. It is not possible to conclude whether or not a person has asthma merely on the basis of data on skin sensitization to aeroallergens, total IgE, eosinophils or lung function tests. Bronchial hyperresponsiveness showed the highest values for sensitivity (97%), PPV (94%), NPV (92%) and accuracy (93%). The second most efficient test is the skin prick test, with PPV 81% and diagnostic accuracy 62%.

SHPS-1 induces aggregation of Ba/F3 pro-B cells via an interaction with CD47.

SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP) substrate-1), a member of the inhibitory-receptor superfamily that is abundantly expressed in macrophages and neural tissue, appears to regulate intracellular signaling events downstream of receptor protein-tyrosine kinases and integrin-extracellular matrix molecule interactions. To investigate the function of SHPS-1 in a hematopoietic cell line, SHPS-1 was expressed in Ba/F3 cells, an IL-3-dependent pro-B-cell line that lacks endogenous SHPS-1 protein. Interestingly, expression of either SHPS-1, or a mutant lacking the intracellular domain of SHPS-1 (DeltaCT SHPS-1), resulted in the rapid formation of macroscopic Ba/F3 cell aggregates. As the integrin-associated protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we investigated whether CD47 played a role in the aggregation of SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate formation was inhibited by an anti-CD47 Ab. Furthermore, erythrocytes from control, but not from CD47-deficient mice, were able to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes do not express integrins, this result suggested that SHPS-1-CD47 interactions can take place in the absence of a CD47-integrin association. We also present evidence that the amino-terminal Ig domain of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47 binding likely triggers bidirectional intracellular signaling processes, these results demonstrate that this interaction can also mediate cell-cell adhesion.

The Src homology 2 (SH2) domain of SH2-containing inositol phosphatase (SHIP) is essential for tyrosine phosphorylation of SHIP, its association with Shc, and its induction of apoptosis.

In this study we have investigated the role that the Src homology 2 domain (SH2) of the 145-kDa 5-phosphatase, SH2-containing inositol phosphatase (SHIP), plays in three of the properties that have been associated with this protein following cytokine stimulation: its association with Shc, its tyrosine phosphorylation, and its inhibition of hemopoietic cell growth. In vitro studies using this SH2 domain revealed that it was capable of binding directly to the Tyr(P)317 motif of Shc with a KD of approximately 290 nM, in keeping with other specific SH2/Tyr(P) interactions. In vivo analysis revealed the SH2 and NPXpY motifs of SHIP acted together, with the Tyr(P)317 and phosphotyrosine binding (PTB) domains of Shc, respectively, to ensure a high affinity SHIP.Shc complex. Expression of cDNAs encoding hemagglutinin-tagged wild type and SH2-inactivated forms of SHIP in the murine hemopoietic cell line DA-ER revealed that wild type SHIP becomes both tyrosine-phosphorylated and associated with Shc following interleukin-3 stimulation, as expected, but the SH2-inactivated SHIPs do neither. Moreover, while the growth rates of parental DA-ER cells and cells expressing these various SHIP constructs are identical, the wild type SHIP-expressing cells die, via programmed cell death, far more rapidly than parental cells. Cells expressing SH2-inactivated SHIPs, on the other hand, show either a reduced or no effect on apoptosis. These results suggest that the SH2 domain of SHIP is required not only for the tyrosine phosphorylation of SHIP and Shc association following cytokine stimulation but also for its induction of apoptosis.

MutS interaction with mismatch and alkylated base containing DNA molecules detected by optical biosensor.

An optical biosensor was used to monitor interactions between the Escherichia coli DNA mismatch repair molecule MutS and various immobilized oligonucleotides. While associating poorly with single-stranded DNA, MutS was capable of rapid association/dissociation from homoduplex DNA. The interaction of MutS with oligonucleotide 30-mers containing single site mismatches demonstrated that during the dissociation phase, MutS binding was greatest to a G-G mismatch, followed by G-T > A-A > C-T, A-C. Binding to A-G, T-T and C-C mispairs was marginally higher than that seen between MutS and homoduplex DNA. The ability of MutS to interact with 30-mers containing alkylated bases was also tested. While binding to O6-methyl-G-C, or to O4-methyl-T-A base pairs was similar to that of homoduplex DNA, strong binding was seen to a O6-methyl-G-T mispair. O4-methyl-T-G, however, was poorly recognized by MutS, with relative binding affinity similar to homoduplex DNA, predicting poor in vivo recognition of O4-methyl-T-G by MutS. Interestingly, MutS demonstrated a relatively high affinity for an 1,N6-etheno-A-T containing homoduplex. Thus, in allowing rapid evaluation of interactions between such molecules, the biosensor will be useful to structure-function analyses.

Changes in microbial populations on fresh cut spinach.

The microbial populations found on fresh-cut spinach leaves that were stored in gas permeable bags at 10 degrees C for 12 days were examined and identified. The microorganisms consisted of mesophilic aerobic bacteria, psychrotrophic bacteria, Pseudomonadaceae, Enterobacteriaceae, Micrococcaceae, lactic acid bacteria and yeasts. Populations of mesophiles, psychrotrophs, Pseudomonadaceae and Enterobacteriaceae increased sharply during the storage period. The initial populations were 10(7), 10(6), 10(6) and 10(4) CFU.g-1 respectively. Populations reached 10(10) for the mesophiles, psychrotrophs and Pseudomonadaceae and 10(7) CFU.g-1 for Enterobacteriaceae after 12 days of storage. Micrococcaceae, lactic acid bacteria and yeasts remained constant (10(3)-10(4) CFU.g-1. The majority of the bacterial isolates were identified as Pseudomonas fluorescens, Aeromonas caviae and Staphylococcus xylosus. The yeasts, which were most frequently isolated, were classified in the genus Cryptococcus. No pathogens such as Listeria monocytogenes and Salmonella were detected. Observations with low temperature scanning electron microscopy (LTSEM) indicated that the microorganisms were not present on the surface of healthy unbroken leaves. Alternatively, they were found in areas where the cuticle was broken and could be seen infecting the internal palisade parenchyma.