Basidiobolus species. Validation of its novelty was established by analysis of the partial ribosomal operon of two isolates from different organs. Phylogeny of ITS and LSU rRNA showed that these isolates belonged to the genus Basidiobolus, positioned closely to B. heterosporus and B. minor. Morphological and physiological data supported the identity of the species, which was named Basidiobolus omanensis, with CBS 146281 as the holotype. The strains showed high minimum inhibitory concentrations (MICs) to fluconazole (>64 µg/mL), itraconazole and voriconazole (>16 µg/mL), anidulafungin and micafungin (>16 µg/mL), but had a low MIC to amphotericin B (1 µg/mL). The pathogenic role of B. omanensis in gastrointestinal disease is discussed. We highlight the crucial role of molecular identification of these rarely encountered opportunistic fungi.Human infectious fungal diseases are increasing, despite improved hygienic conditions. We present a case of gastrointestinal basidiobolomycosis (GIB) in a 20-year-old male with a history of progressively worsening abdominal pain. The causative agent was identified as a novel  Basidiobolus ranarum was reported from Indonesia in 1956 [Basidiobolus species. Infections have also been reported in dogs [B. haptosporus, having 99.97% similarity with the latter species and slightly lower (99.25%) with B. ranarum. Given the low variability of the used marker, these results suggest a possibly undescribed Basidiobolus species.Basidiobolomycosis is a rare infection in humans that usually affects skin and subcutaneous tissues of limbs and trunk. The disorder prevalently occurs in immunocompetent individuals. The first case of subcutaneous basidiobolomycosis caused by  in 1956  describe in dogs . Human g in dogs ,5,6,7. TBasidiobolus has classically been listed as a member of the phylum Zygomycota. On the basis of the molecular phylogeny of small subunit ribosomal RNA, a major taxonomic change has been proposed [Batkoa, Basidiobolus, Conidiobolus, Entomophthora and Zoophthora. Spatafora et al. [proposed . Arbuscuproposed . Acknowlproposed . Using tproposed  raised tproposed  confirmeproposed . Five gea et al.  analysedBasidiobolus and Conidiobolus are sister genera but at large phylogenetic distance, both containing members that are able to cause human infections [Basidiobolus was introduced in the late 19th century [B. haptosporus, B. heterosporus, B. magnus, B. meristosporus, B. microsporus and B. ranarum [B. ranarum [Basidiobolus is a much-debated issue. Unlike many other filamentous fungi, only few phenotypic characteristics are available to differentiate Basidiobolus species. The genus was previously classified in the order Entomophthorales, but was recently assigned to the order Basidiobolales because of its unresolved position close to the flagellated genus Olpidium [fections . Basidio century  and cont century , and als century ,18. Base ranarum . However ranarum . The taxOlpidium .B. haptosporus, having 99.97% similarity with the latter species and slightly lower (99.25%) with B. ranarum. Given the low variability of the used marker, these results suggest that possibly an undescribed Basidiobolus species was concerned. Here, we report the isolation of a similar Basidiobolus species from a human infection in Oman obtained with two strains from different anatomical sites of a single GIB patient. We introduce it as a novel Basidiobolus species, characterised by morphological features and a deviating multilocus sequence profile.The recent report of gastrointestinal basidiobolomycosis from Saudi Arabia  was careFungal strains were isolated from a thrombus that was extracted from the common femoral artery, and from urine. Both strains were sent to the Centraalbureau voor Schimmelcultures (CBS) reference collection housed at the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands and deposited under the accession numbers CBS 146281 and CBS 146282, respectively. The strains were cultured on malt extract agar , potato dextrose agar  and Sabouraud glucose agar . Culture plates were incubated at 25 °C and 30 °C for 2–5 days. Slides were examined with a Nikon Eclipse 80i light microscope, and pictures were taken using a camera attached to the microscope .For molecular identification, DNA was extracted from a fresh culture grown on MEA plates using the cetyltrimethyl ammonium bromide (CTAB) protocol of Möller et al. . The rDNSequence alignments for ITS and LSU regions were generated using MAFFT v7  and analPaecilomyces variotii ATCC 22319, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019) were included as quality controls.Antifungal susceptibility testing (AFST) of CBS 146281 and CBS 146282 was performed using broth microdilution as described in the CLSI document M38-A2. . The folA 20-year-old Omani male with type I diabetes mellitus presented to a local hospital with a two-week history of progressively worsening abdominal pain. A computed tomography (CT) scan of the abdomen revealed a complex cecal mass adherent to the pelvic floor and to the terminal ileum with multiple hepatic lesions. The patient underwent an exploratory laparotomy which revealed cecal perforation, an inflammatory mass at the base of the mesentery of the transverse colon, and extensive cecal, terminal ileum and infrahepatic inflammatory processes. Diverting loop ileostomy and drainage of the pelvic collections were performed and biopsies from multiple sites were taken for histopathological examination and for cultures. Initial histopathological examination of the intraoperative samples demonstrated fungal elements with necrosis and angioinvasion, consistent with mucormycosis. The patient was treated with liposomal amphotericin 7.5 mg/kg/day in combination with caspofungin 50 mg/day. Two days postoperatively, the patient suffered acute ischemia of the right lower limb resulting from an extensive thrombus with total occlusion of the right external iliac artery. He was transferred to Sultan Qaboos University Hospital (SQUH) for further care.Upon presentation at SQUH, physical examination showed a conscious, ill-looking patient with pallor and signs of dehydration. He was afebrile but tachycardic (pulse rate: 104/min) and tachypnoeic (respiratory rate: 22/min) with oxygen saturation of 95% in ambient air. Blood pressure was 117/66 mm Hg. Examination of the abdomen revealed a mildly distended abdomen with midline laparotomy scar, and a functioning ileostomy with a healthy stoma. On palpation, there was right-sided abdominal tenderness with no peritoneal irritation signs. Examination of the lower limbs demonstrated signs of acute ischemia of the right leg with absent femoral, popliteal, pedal and posterior tibial pulses. Other systemic examination was within normal limits. Initial laboratory investigations showed a haemoglobin of 8.9 g/dL, and a total white cell count of 16,700 cells/μL with an eosinophil count of 1000 cells/μL. Biochemistry panel was normal with the exception of alkaline phosphatase of 372 unit/L. Glycosylated haemoglobin was 5.5% . HIV serology was negative.The patient underwent urgent right common femoral artery thrombectomy and embolectomy. Multiplanar CT scan of abdomen and pelvis were performed with oral and intravenous contrast .Then, a limited exploratory laparotomy was performed and entailed right hemicolectomy, drainage of infrahepatic collections and an abdominal wash. Samples from both surgical procedures were sent for histopathology examination and culture. High-dose liposomal amphotericin (7.5 mg/kg/day) in combination with caspofungin 50 mg/day were continued. When empirically treating cases of life-threatening mucormycosis, we use this antifungal combination based on some evidence suggesting synergistic action.Basidiobolus species . In the absence of consensus guidelines or recommendations on the management of basidiobolomycosis, and the lack of antifungal susceptibility results for this particular isolate at this stage, this antifungal combination was chosen based on several case reports describing successful use of azoles and/or amphotericin in treating such infections. The fungal isolate was then sent to the Centre of Expertise in Mycology of Radboud University Medical Centre/Canisius Wilhelmina Hospital, Nijmegen, The Netherlands for susceptibility testing. The isolate was identified by ITS and LSU sequencing as being a novel species within the genus omycetes .Despite the aforementioned surgical interventions and optimisation of antifungal therapy, including the empiric addition of isavuconazole , the disease continued to show fulminant progression, resulting in extensive angioinvasion with involvement of the right common iliac artery, the left infrapopliteal artery and the right renal artery , finallyKlebsiella pneumoniae bacteraemia, ventilator-associated pneumonia and deep surgical site infection, culminating in his death.Furthermore, the hospital course was complicated by severe sepsis secondary to extensively drug-resistant Basidiobolus ranarum (MK501321.1 of strain PHF-MC207) with 95.96% similarity, while LSU sequences matched with Basidiobolus sp.  with 100% similarity. For further understanding of relations between species, phylogenetic analyses were conducted using ITS and LSU sequences, which included available type and reference sequences of Basidiobolus spp. known from culture (Conidiobolus sp. ARSEF 7942), and consisted of 1551 nucleotide positions , of which 1140 were conserved , 402 were variable  and 206 were phylogenetically informative  . Strains CBS 146281 and CBS 146282 clustered in a monophyletic clade, supported by high BS and PP values , and are here described as a novel species, Basidiobolus omanensis. Additionally, the ex-type strain of B. haptosporus var. minor (CBS 310.66) clustered consistently in a well-supported linage, unrelated to the B. haptosporus core clade, and it is therefore elevated to species level below.The analysed sequences of CBS 146281 and CBS 146282 were found to be identical. BLAST searches of ITS sequences in GenBank matched with those of  culture . Each daU = 109) . PhylogeB. omanensis was performed using broth microdilution protocols according to CLSI M38A2, resulting in the following MICs: amphotericin B 1 µg/mL; fluconazole >64 µg/mL, itraconazole and voriconazole >16 µg/mL; and anidulafungin and micafungin >16 µg/mL.Antifungal susceptibility testing of Basidiobolus omanensis Al-Hatmi, Sand.-Den., Balkhair, Al Adawi & de Hoog, sp. nov.—Etymology: ‘omanensis’ refers to the country in which the fungus was first isolated.Holotype: Oman, Muscat, from thrombus in common femoral artery of human patient, CBS 146281, designated here, preserved in metabolically inactive condition in liquid nitrogen. Living strain derived from type CBS 146281.Description: based on colonies of CBS 146281 grown for 5 d at 25 °C on MEA and SGA. On MEA, colonies grow rapidly, with an average daily growth rate of 5–6 mm; whitish, flat, waxy, glabrous and radially folded; margin entire, straight A,B. On SHyphal elements hyaline, unbranched, coenocytic or with occasional septa, broad, 5–10 μm in diameter (2 to 4) I,J. PrimBasidiobolus minor (Sriniv. & Thirum.) Al-Hatmi, Sand.-Den. & de Hoog, comb. et stat. nov.—MycoBank MB 839873.Basionym: Basidiobolus haptosporus var. minor Sriniv. & Thirum., Mycopath. Mycol. Appl. 33: 60. 1967.Basidiobolus species causing refractory angioinvasive gastrointestinal disease (GIB) in a 20-year-old Omani patient with type 1 diabetes mellitus. His disease seemed to have started in the caecum with extension to the major vessels of the lower limbs. Physical examination of the lower limbs demonstrated signs of acute ischemia, confirmed by imaging studies including CT  of 16,700 cells/µL, with an elevated eosinophil count of 1000 cells/µL, normal glycosylated haemoglobin and deranged liver function tests. These results agreed with previously reported cases where elevated WBCs and eosinophilia were observed . Abdominstandard . From th conidia . The thi conidia F. Overalferences .Basidiobolus ranarum with 95.96% similarity, while LSU sequences matched 100% with an unnamed Basidiobolus species. The large ITS distance to a described species, and judging from the phylogenetic ribosomal tree, led to the conclusion that our isolate is taxonomically separate from the recognised species of Basidiobolus , a species which is commonly linked to GIB [B. omanensis isolates as a novel species within the genus Basidiobolus; the differences observed for all phylogenetic markers are considered sufficient to propose B. omanensis as a new species of Basidiobolus. The phenotypic characteristics proposed in the literature provide insufficient resolution to differentiate the seven known species of Basidiobolus; these characters mostly include odour production along with physiological features [B. omanensis is similar to remaining species in terms of colonies with satellites, conidia, conidiophores, zygospores and chlamydospores.BLAST searches of the rDNA sequences of CBS 146281 and CBS 146282 revealed that the ITS sequences matched a strain of diobolus  and that ranarum . A previ lineage . Accordifeatures ,48,49. H


There are more than 30 inherited human disorders connected with repeat expansion . Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear.An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures.There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structurescan block various stages of DNA metabolism processes, such as replication, repair and recombination and it isconsidered as the source of repeat instability. However, none of the hypotheses is fully confirmed or is the onlyvalid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription andTCR-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of(CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNAmetabolism and, therefore, the developed systems are universal and applicable for various studies. Transgeniccell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantification was developed. One type of the cell lines contains anexogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vectoris maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for finding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed forthe quantification of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome. Repeat expansion is a unique type of mutation that is characterized by a dramatic increase of the number of triplet repeatsin DNA. Triplet repeats are more prone to expansion: to date,more than 30 diseases associated with their instability areknown . Fragile X syndrome, asthe most common form of hereditary intellectual disabilityis also based on triplet repeat expansion. The cause of thedisease is the expansion of the CGG repeat located in the5′-untranslated region of the FMR1 gene. Normally, the repeatsnumber is relatively stable and does not exceed 54 triplets; ifthe (CGG)n expansion increases up to 200 triplets, the FMR1allele becomes premutant, and the ataxia/tremor syndromesand primary ovarian insufficiency syndrome associated with aFragile X syndrome develop. The premutant allele frequencyin the population is 1 : 100. Even though the clinical manifestations are often not observed, the expanded repeat can betransmitted over generations. Full mutation develops whentriplets numbers increase over 200: the FMR1 gene promoterbecomes methylated, the locus is heterochromatinized, and theFMRP protein is completely lost, which leads to the development of Fragile X syndrome. FMRP is necessary for normalneuron activity and its absence causes pronounced phenotypicmanifestations: macroorchidism, endocrine pathologies, cerebellum morphological changes, and intellectual disabilitycharacterized by behavior and learning problems . The fullmutation frequency varies from 1 : 4,000 in men, and up to1 : 6,000 in womenDespite understanding the syndrome pathogenesis details,the expansion mechanism has not yet been studied. Differentprocesses of DNA metabolism are probably able to increasethe CGG-repeat instability. Therefore, the contribution ofreplication to the expansion processes has been established:the formed hairpin on the newly synthesized DNA strandleads to the additional replication of the region containing the(CGG)n repeat and, therefore, to its increasing . However, in people suffering from repeat expansiondisorders, and in model mice, expansion is also observed intissues with low proliferative activity, including the brainlobes, oocytes, liver and muscles ; itconfirms the theory that the repeat expansion can also dependon other processes affecting DNA. Indeed, for many proteinsof the DNA repair and recombination pathways, their probable participation in the repeat expansion process has beenshown. Some experimental data indicate the MMR systemcomponents involvement in the expansion . Another possible source of instability can be transcription and transcription-coupled repair(TCR-NER), since many repeat tracts are characterized by theR-loops formation – RNA: DNA-resistant duplexes formingduring RNA synthesis as well as the disruption of the initiation of PolII transcription . Thelesions during transcription initiate TCR, a form of excisionalnucleotide repair (NER). For some proteins of this cascade,correlations with the (CGG)n instability level were found.Itshould be noted that for the FMR1 premutant alleles, whichrapidly accumulate repeated units, a significant increase of theFMR1 transcription level was found, which probably indicatesthe involvement of the TCR system in the repeat instabilitydevelopment. However, there is no unequivocal confirmationof this hypothesis.To study the details of all the described cascades, it isnecessary to have a model in which it is possible to track allthe changes occurring with repeat and surrounding regionsin response to the induction of a certain DNA metabolismprocess. To date, similar models have already been proposed, but noneof them can directly assess the contribution of transcription to(CGG)n instability. In this study, we describe the experimental models for repeat instability research based on two typesof plasmids: integrated and not integrated into the genome.These models will allow taking into account the contributionof replication, transcription, TCR-NER, and genome locationto the CGG-repeat instability. In addition, this model can beused to study the repeat-induced mutagenesis observed incells with an expanded repeat in the FMR1 promoter region.Ethics statements. The procedure of involving the patientsin the study was strictly designed in accordance with international standards, which include the awareness of the subject,their consent to participate in the study in its entirety, andthe guarantee of confidentiality. All of the studies conformedto the ethical standards developed in accordance with theHelsinki Declaration of the World Medical Association asamended in 2000. In addition, the studies were supervisedby the Institutional Review Board. The written consent of thestudy participants was also obtained.DNA purification and repeat sizing. Peripheral venousblood from all of the patients was collected in NovosibirskCity Clinical Hospital No. 1 into EDTA-containing tubes andfrozen before DNA purification. The DNA was purified fromwhole venous blood and cell cultures using a Wizard® Genomic DNA Purification Kit . CGG repeats were sized using a special protocol for theGC-rich DNA amplification proposed earlier . For PCR primers NewFraxC (5′-d6RG-tgctttctagactcagctccgtttcggtttcacttccggt-3′) and NewFraxR4 (5′-taagcagaattcccttgtagaaagcgccattggagccccgca-3′) and 0.02 unitsof Q5-DNA polymerase were used. The resulting fragmentswas separated by agarose gel electrophoresis. To assess theaccurate size of the repeat, capillary electrophoresis using a1200 LIZ length standard  wasperformed. The flanking region in the PCR product is a totalof 269 bp, thereby the repeat length was determined by thefollowing equationCloning CGG repeats of various lengths into vectorsystems. The control plasmid pCDH containing no CGG-repeat consisted of the following elements: (1) doxycyclineinducible Tet-O-minimal CMV promoter, IRES sequence,open reading frame (ORF) of the GFP protein, (2) constitutive promoter EF1alpha, transactivator for Tet-O-elementrTtA ORF, T2A peptide, the DsRedExpress protein ORF,(3) beta-lactamase promoter, beta-lactamase protein ORF fortransformed bacterial cells selection, origin of replication, and(4) SV40 origin of replication. The PCR product carrying theCGG repeat was cloned into the pCDH plasmid at the XbaIand EcoRI restriction endonuclease sites between the CMV minimal promoter and the IRES sequence.Plasmid pSBi for CGG-repeat cloning was assembled fromthe following components: (1) beta-lactamase promoter, betalactamase protein ORF for transformed bacterial cells selection, origin of replication, (2) Sleeping Beauty transposonterminal repeats, (3) cassette containing a PGK promoterand a puromycin-N-acetyl transferase ORF, (4) an hPGKpromoter, an rTta ORF, (5) an inducible TRE3GS promoter,and an mGFP ORF. Cloning of the PCR product containing CGG-repeat drivenby an inducible promoter was also carried out at the restriction endonuclease sites XbaI and EcoRI (SibEnzyme). For theplasmids production, the electrocompetent cells of the E. colistrain NebStable  were transformed. It was shown that an extended repeat during the transformation of bacterialcells and their cultivation is prone to a dramatically repeatlength contraction, which is consistent with the literaturedata ; therefore, the NebStable cells werecultured for a day at 20 °C to avoid the repeat size decrease.For HEK293A and HEK293T cells transfection, plasmidswere isolated and purified using the QIAGEN® Plasmid PlusMaxi Kit .Eukaryotic cell transfection. HEK293A and HEK293Tcells transfection was performed using the Lipofectamine3000 reagent . The inductionof the Tet-O-minimal CMV promoter and TRE3GS promoterwas performed using the doxycycline with a concentration of1 μg/ml in the cultural media.Assembly of experimental plasmidscarrying CGG-repeat of normal and premutant lengthsWe obtained a set of plasmids based on eukaryotic expression vectors with an inducible promoter that regulates theCGG-repeat transcription level of CGG repeat of normal orpremutant length and GFP ORF. These plasmids serve as thecore of the model system for studying (CGG)n repeat instability. The pCDH plasmid was used as a vector for transientexpression and exogenous CGG-repeat maintenance in a nonintegrated state in the genome . For the integrationof the exogenous CGG-repeat into the genome, a constructbased on the Sleeping Beauty pSBi transposon/transposasesystem was assembled .The pCDH plasmid encodes two reporter proteins:DsRedExpress driven by EF1 promoter and EGFP, whoseexpression is regulated by the inducible Tet-O-CMV promoter.Downstream of the Tet-O-CMV promoter, a multiple cloningsite for CGG-repeat cloning is located. Due to this mutual arrangement of the inducible promoter and the site of the repeatcloning, it can be established that transcription goes throughthe inserted CGG-repeat due to the synthesis of EGFP mRNA.After several transcription rounds, the influence of transcription on the repeat instability can be detected. In addition,pCDH plasmid contains the SV40 origin of replication andthereby it is able to replicate in HEK293T cells that producethe SV40 large T antigen. In this case, it is possible to assessnot only the contribution of transcription, but also the role ofreplication processes during the maintenance of the pCDH inthe form of an episome. The pSBi vector encodes an mGFP protein driven by aninducible TRE3GS promoter. Before the mGFP ORF are sitesfor cloning the CGG repeat. Therefore, it is possible to analyze the effect of transcription on changes in the CGG repeatlength. Since this vector is based on a transposon, a part ofthe plasmid flanked by specific repeated sequences recognizedby SB transposase and this part of plasmid can be insertedinto different regions of the genome by transposase. It is possible to assess the potential influence of the integration siteson the instability of the CGG repeat by the determination ofthe insertion sites.To obtain fragments containing a CGG-repeat, we usedDNA samples isolated from continuous human B-lymphocytes cultures and whole blood samples of patients with Fragile Xsyndrome .To create a construct with exogenous CGG-repeat it wasdecided to use repeats of normal and premutant lengths. It isexpected that the instability of these types of repeats will besignificantly different, since the premutant allele is the mostunstable, and the normal allele, on the contrary, is prone toonly insignificant polymorphism .As a result, five types of plasmids were obtained. Theseplasmids carry 5 (pCDH-5), 25 (pSBi-25), 59 (pCDH59),85 (pCDH85), and 160 repeats (pSBi-160), respectively. Thestructures of all plasmids were confirmed by Sanger sequencing .Study of the experimental plasmid functionality The eukaryotic cells transfection efficiency by the assembledconstructs was evaluated to confirm the correct expressionof reporter proteins in the presence of an extended repeat(CGG)n. It has been shown that transfection and reporter protein synthesis after transfections by plasmids carrying CGGrepeat of normal or premutant length occurs with the sameefficiency as transfection with control plasmids (without therepeated sequence). When cells were transfected with pCDHplasmids, the expression of DsRedExpress was observed.It was also possible to carry out selection on a medium with thepuromycin of cells transfected with pSBi vectors. The abilityof the tetracycline/doxycycline-inducible promoters regulating green fluorescent protein expression to spontaneous activationwas investigated. It is important that the transgenic cells donot have a background EGFP expression because spontaneouspromoter activation can interfere with the accurate assessmentof the CGG-repeat instability level during transcription andtranscription-coupled repair Cells transfected with pCDH the showed active expression of the red protein (driven by constitutive promoter EF1)and the absence of the green protein expression (regulatedby inducible promoter) without the promoter induction. Forinduction, doxycycline was added daily to the cells, resultingin a high level of green protein fluorescence . When plasmid pSBi containing the TRE3GS promoter was used, nobackground induction was observed. It allows for performinga selection using puromycin to obtain stable transformants andto avoid the background transcription level influence on theinserted CGG-repeat .Development of the methodfor analyzing repeat instability in model cell linesBy using the obtained transgenic cell lines, we expect thatthe expansion in different cells of the culture will occur atdifferent rates, and, as a result, we will receive a mosaic culture. In this regard, it is necessary to use a value allowing the grading of somatic instability and allowing the comparison ofcell lines carrying different alleles of exogenous CGG-repeat.Previously, different approaches were proposed to assessthe trinucleotide repeats somatic instability in patients withrepeats expansion disorders. For example, the method for theassessment of CAG-repeat instability in Huntington’s diseaseis based on the main allele determination by the maximumpeak as a result of fragment analysis and additional peaks, followed by normalization to the summed values of the heightsof all peaks . Another method for the repeatinstability level assessment is based on the serial dilution ofthe template followed by PCR – small-pool PCR .Amplification by dilution enables the detection of mosaicism, which cannot be detected by conventional PCR due tothe low synthesis efficiency of less represented or very largealleles. However, these methods are insufficiently applicableto assess the instability (CGG)n, since the amplification of thelarger allele occurs with much less efficiency than the shorterallele synthesis . To quantify the CGG-repeat instability in developed cell lines, we proposed an analysis methodbased on the calculation of the somatic instability index (ISI)after the amplification of GC-rich templates according to themethod of B.E. Hayward et al. (2016). This value enables oneto take into account not only the repeat size but also the spreadof values between alleles, regardless of the efficiency of theirsynthesis. For (CGG)n repeats located in the FMR1 gene, wepropose the calculation of ISI using the following equationThe median is a value separating the raw data into twohalves, and it considers the number of alleles. This valuetakes into account sample heterogeneity, and not sensitive tothe detection of repeat lengths that are too long or too short,unlike using an arithmetic mean. When using the arithmeticmean in the index calculating, the contribution of larger alleles will be taken into account more than the contribution ofshorter ones. As a result, cell lines with different degrees ofthe exogenous repeat instability can have similar values ofthe magnitude of somatic instability, which will lead to thefalse results interpretation. The value (Nmax – Me) takes intoaccount the diversity and scatter of values in samples, wherelarge Me values indicate a large median repeat length. TheISI calculation does not take into account the amount of PCRproduct (peak height) for each allele, i. e. PCR efficiency doesnot affect the final value. To determine the somatic instabilityindex, the DNA of eleven patients with Fragile X syndromewas isolated from whole blood, which served as a startingmaterial for the synthesis of extended repeats (CGG)n .As can be seen from the calculation of the ISI , index increases with an increase in the number and spread of repeatvalues. It should be noted that the method of analysis of instability works for two or more alleles in patients with mosaicism. In the case of one allele, we take the index of somaticinstability equal to the size of the CGG-repeat, since the patientwith one allele has Nmax – Me = 0. We cannot accept ISI = 0because the CGG-repeat is unstable by nature.Fragile X syndrome is one of the most common causes ofhereditary intellectual disability . Thefrequency of full mutation in the human population varies from1 : 6,000 in women to 1 : 4,000 in men, while the premutantallele, as the most unstable allele of the FMR1 gene promoterregion, occurs in 1 : 100 cases. The instability of the CGGrepeat is expressed in its tendency toward expansion – a multiple and rapid increase of the tract repeated sequence length.In addition, in the cells and tissues of patients as well as thetissues of model animals, repeat contractions are observed,that lead to somatic mosaicism and its degree correlates withthe severity of symptoms . However, theprobability of expansion is in tenfold higher than contraction, whichmay be the reason for the increased severity of the diseasesmanifestations during transmission in a number of generationsThere are a number of hypotheses explaining the expansionmechanism, but none of them have been sufficiently supportedby experimental data. All of the hypotheses – assume as amain reason for repeat instability – the formation of alternativeDNA secondary structures at a certain site in DNA during thedifferent processes of DNA metabolism, which can disrupt these processes. In vitro and in vivo experiments have shownthe formation of alternative DNA secondary structures, suchas hairpins, R-loops, and G-quadruplexes . Such structurescan significantly violate these processes of DNA metabolism,which in turn affects the instability of repeats. One of the possible reasons is associated with the slippage of a DNA strandduring replication . Today, it is absolutely clear that the slippage of DNAstrands can occur in various cases: during DNA replicationin dividing cells as well as during repair processes. However,this model cannot reliably explain why not all of the repeatsexpand or and why the threshold value for the length of therepeat sequence is similar for different diseases. There is evidence for the contribution of some repair cascade proteins,which are proteins that are necessary for recombination andtranscription to repeat instability. However, all of the hypotheses have certain drawbacks and contradictions; therefore,it is necessary to continue the search for the molecular mechanism of repeat expansion.An expansion model based on a transgenic cell line containing exogenous (CGG)n repeat can serve as a convenientexperimental system. In such systems it is possible to trackchanges in repeat length in response to the induction of different cascades of DNA metabolism. Using the transgeniccell lines obtained in this study make it possible to assess thecontribution of replication, transcription, and repair in thecell to CGG-repeat instability. We have assembled two typesof plasmids: based on the SV40 origin plasmid, capable ofreplicating in cell cultures expressing the SV40 T antigen,and based on the Sleeping Beauty transposon-based vectorsystem for integrating the cassette with CGG-repeat andreporter proteins into various genomic loci. The transfectionefficiency and the initial expression level of reporter proteinswere comparable to those of the control plasmid withoutthe (CGG)n repeat. It is also possible to obtain a transgeniccell culture with single genotype using different approachessuch as sorting or limiting dilutions with antibiotic selection.Changes in the length of an exogenous repeat and, therefore,mosaicism that will take place in transgenic culture over timecan be detected and estimated using the developed ISI index.This method is useable and reflects the correlation betweenrepeat instability and phenotypic manifestations of the diseasesobserved in the different brain lobes of patients with Fragile Xsyndrome and associated disorders.In the created experimental cell lines it is possible to directlyassess the level of repeat expansion or contraction as well asthe changes caused by repeat instability. The design of vectorsystems makes it possible to detect changes in the length ofthe exogenous CGG-repeat at different genome loci, duringcultivation for a long or short time, with or without promoterinduction. Measuring the fluorescent proteins expressionlevels can serve as basis for tracking the possible increase ofinstability and mutations accumulation mediated by repeatinduced mutagenesis. To determine the contribution of specificproteins from various cascades to the development of instability, it is possible to carry out chromatin immunoprecipitationusing transformed cells. In addition, the level of instability inthe created cellular models of CGG-repeat expansion can beassessed by the proposed index of somatic instability. Index ISI should also have a biological meaning, i. e. reflect the degreeof phenotypic changes in patients with Fragile X associateddisorders. To test this hypothesis, a study of the dependencesof ISI values in patients with changes in the brain accordingto FMRI data was started. The preliminary data indicate somecorrelations, but more research is needed.To date, the mechanism of the instability of trinucleotiderepeats remains not fully understood. At the same time, thisresearch area remains extremely urgent due to the fact thatthe diseases caused by this mutation are socially significant.To search for the repeat instability reasons, it is necessary todevelop cellular models for tracking all of the changes causedby expansion, as well as to evaluate the contribution of variousproteins and DNA metabolism pathways to this process. Theconstructs developed in this work for instability assessing canbe used in such studies. Various cell lines can be transfected with the assembledplasmids. We tested the efficiency of the constructs in two celllines: HEK293A and HEK293T. After cell transfection and theinduction of reporter protein expression, at various passages,it is possible to accurately determine the repeat size (CGG)n,as well as other parameters and show the presence or absenceof CGG-repeat expansion, depending on its initial length andthe number of passages. In the future, our model can be usedin studies for the determination of all the aspects of repeatinstability in the human genome and it will help form a morecomplete understanding of the mechanisms of this mutation.The authors declare no conflict of interest.Bontekoe C.J.M. Instability of a (CGG)98 repeat in the Fmr1 promoter. Hum. Mol. Genet. 2001;10(16):1693-1699. DOI 10.1093/hmg/10.16.1693.DeJesus-Hernandez M., Mackenzie I.R., Boeve B.F., Boxer A.L.,Baker M., Rutherford N.J., Nicholson A.M., Finch N.A., Flynn H.,Adamson J., Kouri N., Wojtas A., Sengdy P., Hsiung G.Y.R., Karydas A., Seeley W.W., Josephs K.A., Coppola G., Geschwind D.H.,Wszolek Z.K., Feldman H., Knopman D.S., Petersen R.C., Miller B.L., Dickson D.W., Boylan K.B., Graff-Radford N.R., Rademakers R. Expanded GGGGCC hexanucleotide repeat in noncodingregion of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011;72(2):245-256. DOI 10.1016/j.neuron.2011.09.011.Fouche N., Ozgur S., Roy D., Griffith J.D. Replication fork regressionin repetitive DNAs. Nucleic Acids Res. 2006;34(20):6044-6050.DOI 10.1093/nar/gkl757.Gorbunova V., Seluanov A., Dion V., Sandor Z., Meservy J.L., Wilson J.H. Selectable system for monitoring the instability of CTG/CAG triplet repeats in mammalian cells. Mol. Cell. Biol. 2003;23(13):4485-4493. DOI 10.1128/mcb.23.13.4485-4493.2003.Grishchenko I.V., Purvinsh Y.V., Yudkin D.V. Mystery of expansion:DNA metabolism and unstable repeats. In: Zharkov D.O. (Ed.).Mechanisms of Genome Protection and Repair. Cham: SpringerInternational Publishing, 2020;101-124. DOI 10.1007/978-3-030-41283-8_7.Groh M., Lufino M.M.P., Wade-Martins R., Gromak N. R-loops associated with triplet repeat expansions promote gene silencing inFriedreich ataxia and fragile X syndrome. PLoS Genet. 2014;10(5):e1004318. DOI 10.1371/journal.pgen.1004318.Hayward B.E., Zhou Y., Kumari D., Usdin K. A Set of assays for thecomprehensive analysis of FMR1 alleles in the Fragile X-related disorders. J. Mol. Diagn. 2016;18(5):762-774. DOI 10.1016/j.jmoldx.2016.06.001.Heulens I., Suttie M., Postnov A., De Clerck N., Perrotta C.S., Mattina T., Faravelli F., Forzano F., Kooy R.F., Hammond P. Craniofacial characteristics of fragile X syndrome in mouse and man. Eur. J.Hum. Genet. 2013;21(8):816-823. DOI 10.1038/ejhg.2012.265.Jensen M.A., Fukushima M., Davis R.W. DMSO and betaine greatlyimprove amplification of GC-rich constructs in de novo synthesis.PLoS One. 2010;5:e11024. DOI 10.1371/journal.pone.0011024.Kononenko A.V., Ebersole T., Mirkin S.M. Experimental system tostudy instability of (CGG)n repeats in cultured mammalian cells.In: Richard G.-F. (Ed.). Trinucleotide Repeats: Methods and Protocols. New York: Springer, 2020;137-150. DOI 10.1007/978-1-4939-9784-8_9.Kovalenko M., Dragileva E., St Claire J., Gillis T., Guide J.R., New J.,Dong H., Kucherlapati R., Kucherlapati M.H., Ehrlich M.E.,Lee J.M., Wheeler V.C. Msh2 acts in medium-spiny striatal neuronsas an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington’s disease knock-in mice. PloS One. 2012;7(9):e44273. DOI 10.1371/journal.pone.0044273.Krasilnikova M.M., Kireeva M.L., Petrovic V., Knijnikova N., Kashlev M., Mirkin S.M. Effects of Friedreich’s ataxia (GAA)n*(TTC)nrepeats on RNA synthesis and stability. Nucleic Acids Res. 2007;35(4):1075-1084. DOI 10.1093/nar/gkl1140.Lam E.Y.N., Beraldi D., Tannahill D., Balasubramanian S. G-quadruplex structures are stable and detectable in human genomic DNA.Nat. Commun. 2014;4(1)1-8. DOI 10.1038/ncomms2792.Lee J.M., Zhang J., Su A.I., Walker J.R., Wiltshire T., Kang K., Dragileva E., Gillis T., Lopez E.T., Boily M.J., Cyr M., Kohane I., Gusella J.F., MacDonald M.E., Wheeler V.C. A novel approach to investigate tissue-specific trinucleotide repeat instability. BMC Syst.Biol. 2010;4(1):29. DOI 10.1186/1752-0509-4-29.Lokanga R.A., Entezam A., Kumari D., Yudkin D., Qin M., Smith C.B.,Usdin K. Somatic expansion in mouse and human carriers of fragile X premutation alleles. Hum. Mutat. 2013;34(1):157-166. DOI10.1002/humu.22177.Mailick M.R., Movaghar A., Hong J., Greenberg J.S., DaWalt L.S.,Zhou L., Jackson J., Rathouz P.J., Baker M.W., Brilliant M., Page D.,Berry-Kravis E. Health profiles of mosaic versus non-mosaic FMR1premutation carrier mothers of children with fragile X syndrome.Front. Genet. 2018;9:173. DOI 10.3389/fgene.2018.00173.Martin G.E., Roberts J.E., Helm-Estabrooks N., Sideris J., Vanderbilt J., Moskowitz L. Perseveration in the connected speech of boyswith fragile X syndrome with and without autism spectrum disorder.Am. J. Intellect. Dev. Disab. 2012;117(5):384-399. DOI 10.1352/1944-7558-117.5.384.Monckton D.G., Wong L.J.C., Ashizawa T., Caskey C.T. Somatic mosaicism, germline expansions, germline reversions and intergenerational reductions in myotonic dystrophy males: small pool PCRanalyses. Hum. Mol. Genet. 1995;4(1):1-8. DOI 10.1093/hmg/4.1.1.Morales F., Couto J.M., Higham C.F., Hogg G., Cuenca P., Braida C.,Wilson R.H., Adam B., Del Valle G., Brian R., Sittenfeld M., Ashizawa T., Wilcox A., Wilcox D.E., Monckton D.G. Somatic instabilityof the expanded CTG triplet repeat in myotonic dystrophy type 1 isa heritable quantitative trait and modifier of disease severity. Hum.Mol. Genet. 2012;21(16):3558-3567. DOI 10.1093/hmg/dds185.Pearson C.E., Sinden R.R. Alternative structures in duplex DNAformed within the trinucleotide repeats of the myotonic dystrophyand fragile X loci. Biochemistry. 1996;35(15):5041-5053. DOI10.1021/bi9601013.Roberts J., Hennon E.A., Anderson K. Fragile X syndrome and speechand language. ASHA Leader. 2003;8(19):6-27. DOI 10.1044/leader.FTR2.08192003.6.Shah K.A., Mirkin S.M. The hidden side of unstable DNA repeats:Mutagenesis at a distance. DNA Repair. 2015;32:106-112. DOI10.1016/j.dnarep.2015.04.020.Usdin K., Woodford K.J. CGG repeats associated with DNA instabilityand chromosome fragility form structures that block DNA synthesisin vitro. Nucleic Acids Res. 1995;23(20):4202-4209.Woodford K., Weitzmann M.N., Usdin K. The use of K(+)-free buffers eliminates a common cause of premature chain termination inPCR and PCR sequencing. Nucleic Acids Res. 1995;23(3):539. DOI10.1093/nar/23.3.539.Yudkin D.V., Lemskaya N.A., Grischenko I.V., Dolskiy A.A. Chromatin changes caused by expansion of CGG repeats in fmr1 gene. Mol.Biol. 2015;49(2):179-184.Zhao X.-N., Lokanga R., Allette K., Gazy I., Wu D., Usdin K.A MutSbeta-dependent contribution of MutSalpha to repeat expansions in fragile X premutation mice? PLoS Genet. 2016;12(7):e1006190. DOI 10.1371/journal.pgen.1006190.


Candidemia patients treated at Siriraj Hospital  during January 2016 to December 2017 were enrolled. A total of 156 patients  were included. The most prevalent underlying conditions were diabetes (32.1%), chronic cardiac disease (28.2%), chronic kidney disease (26.9%), and hematologic malignancies (21.2%). Candida species isolated from patient blood were C. tropicalis (49.4%), C. albicans (28.8%), C. glabrata (16.7%), and C. parapsilosis (5.1%). Fluconazole resistance was significantly increased in C. tropicalis (37.8%). No independent risk factors were associated with patients with non-albicans Candida candidemia compared to those with C. albicans candidemia. There was no significant difference in mortality between patients with non-albicans Candida candidemia and patients with C. albicans candidemia . When compared with C. albicans candidemia, multivariate analysis revealed chronic liver disease , neutropenia , and male gender  to be independent risk factors for C. tropicalis candidemia. The observed high resistance of C. tropicalis to fluconazole indicates that fluconazole should not be used for empirical antifungal treatment in these patients.This study aimed to investigate the risk factors for and the outcomes of patients with candidemia caused by non- Candida spp. is the most common fungal pathogen that causes nosocomial infection [Candida, is a serious public health problem that is associated with high mortality and high medical and economic costs [Candida infection increases following advanced medical interventions, such as hematopoietic stem cell transplantation and solid organ transplantation, as well as after invasive medical procedures, such as the use of vascular catheter or mechanical ventilator [nfection ,2. Candiic costs , and theic costs ,5. The intilator . Factorsalbicans Candida has risen dramatically in some countries, especially in Asia, and there has been a shift from Candida albicans to non-albicans Candida species as the more prevalent causative pathogen in candidemia patients [Candida tropicalis is the most common non-albicans Candida species in tropical countries, including Thailand [Candida glabrata [C. tropicalis isolates exhibit decreased susceptibility to azole agents [Candida species [albicans Candida is relatively higher than the mortality rate in candidemia caused by C. albicans [Previous epidemiological studies demonstrated the same trend toward increasing numbers of patients with candidemia in both neutropenic and non-neutropenic individuals. The incidence of non-patients ,9,10,11.Thailand . Althougglabrata , C. trope agents ,14. The  species . Howeveralbicans . Early aalbicans . Delay ialbicans .albicans Candida are still unclear. Accordingly, the aim of this study was to identify the independent risk factors for candidemia caused by non-albicans Candida, particularly C. tropicalis. The secondary objective was to investigate antifungal susceptibility and mortality among non-albicans Candida candidemia patients.Despite the existing data from previous studies, the risk factors and clinical outcomes of patients with bloodstream infection from non-This retrospective cohort study included candidemia patients diagnosed and treated at the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand during January 2016 to December 2017. Siriraj Hospital is a 2300-bed national super-tertiary referral center. This study was registered in the Thai Clinical Trials Registry (ID: TCTR20190501003).Candida spp. were enrolled. Patients whose blood culture grew more than 1 species of Candida, who had recurrent candidemia, and/or who had an incomplete medical record were excluded. All candidemia patients aged 18 years or older who had a positive blood culture for 3, yeast in non-sterile body samples, prior antifungal exposure, and comorbidities. Candidemia was categorized into primary fungemia, acute disseminated candidiasis, catheter-related blood stream infection, intra-abdominal infection, hepatosplenic abscess, bone and joint infection, central nervous system infection, endocarditis, and urinary tract infection. All Candida isolated from blood cultures were identified to the species level. The species identification of Candida was performed based on biochemical characteristics using chromogenic medium (Brilliance™ Candida agar) and RapID™ Yeast Plus Panel . Antifungal susceptibility was tested by the microbroth dilution method using Thermo Scientific™ Sensititre™ YeastOne™ YO9 AST Plate . Minimal inhibitory concentration (MIC) breakpoints were interpreted according to the Clinical Laboratory Standards Institute (CLSI). Antifungal susceptibility test results and clinical outcomes were analyzed. Collected patient demographic and clinical data included age; gender; duration of hospital stay; and risk factors for candidemia, including central venous catheter, mechanical ventilation, parenteral nutrition, hemodialysis, peritoneal dialysis, recent abdominal surgery, ICU admission, urinary catheter, prosthesis, broad spectrum antibiotics, corticosteroid equivalent to prednisolone at least 20 mg/day for at least 2 weeks, absolute neutrophil count < 500 cells/mmt-test was used to compare normally distributed continuous data, and the Mann–Whitney U test was used to compare non-normally distributed continuous data. Multivariate analysis via multiple logistic regression model (backward stepwise) included all variables with a p-value less than 0.2 from univariate analysis. All statistical analyses were performed using SPSS Statistics (v18.0) software , and a 2-sided p-value less than 0.05 was considered to be statistically significant.Continuous data are presented as mean ± standard deviation (SD) for normally distributed data, and as median and interquartile range (IQR) for non-normally distributed data. Categorical data are presented as frequency and percentage. The Chi-square test or Fisher’s exact test was used for the comparison of categorical variables between two groups. The independent A total of 156 patients were enrolled. The mean age was 64 years, and 56.4% of patients were male. The most prevalent underlying conditions were diabetes mellitus (32.1%), chronic cardiac disease (28.2%), chronic kidney disease (26.9%), and hematologic malignancies (21.2%) .Candida species isolated from patient blood were C. tropicalis (49.4%), C. albicans (28.8%), C. glabrata (16.7%), and C. parapsilosis (5.1%)  than in C. albicans (14.3%) (The s (5.1%) . Antifun (14.3%) .albicans Candida and C. albicans candidemia are shown in albicans Candida candidemia in univariate analysis.Patient characteristics and other factors significantly associated with non-C. tropicalis or C. albicans to identify the factors associated with those two infections is shown in C. tropicalis infection , which is consistent with a previous study in Thailand, and which reveals an increasing incidence of non-albicans candidemia [C. tropicalis was found to be the most common Candida species in patients with candidemia, and this is similar to the results of previous studies conducted at the same hospital [C. tropicalis may be associated with a warmer climate [Candida species in our study.From January 2016 to December 2017, we analyzed data from 156 candidemia patients admitted to Siriraj Hospital, the largest university hospital in Thailand. Most patients had candidemia that was caused by non-ndidemia . C. trophospital . It was  climate , which malbicans Candida candidemia. A previous study also reported non-albicans Candida candidemia to be associated with neutropenic patients [albicans Candida candidemia.The present study identified neutropenia and chronic liver disease as potential risk factors for non-patients . HoweverC. tropicalis and C. albicans candidemia were also analyzed in this study. The results of that analysis revealed neutropenia, chronic liver disease, and male gender to be independent risk factors for C. tropicalis candidemia. A previous study reported the independent risk factors for C. tropicalis candidemia to be abdomen portal entry, hematologic malignancies, and older age [C. tropicalis seems to originate from the intra-abdominal origin, and it tends to infect neutropenic patients.Risk factors associated with lder age . Our stuCandida tropicalis has become increasingly resistant to fluconazole. Two previous studies that were conducted at our center in 2009 and 2013 found rates of C. tropicalis susceptibility to fluconazole of 100% and 70.3%, respectively [C. tropicalis susceptibility to fluconazole of only 62%. Therefore, empirical antifungal treatment with echinocandins or amphotericin B product in candidemia patients with risk factors for C. tropicalis infection is strongly recommended. In our study, most of the patients infected with fluconazole-resistant C. tropicalis did not have a history of azole exposure and they may be acquired from the environment [Candida and environmental azole exposure needs to be investigated.ectively ,19. Howeironment . Azoles C. albicans and non-albicans Candida candidemia. This extremely high mortality rate in candidemia patients highlights the need to improve candidemia management guidelines in Thailand.The mortality rate among candidemia patients in this study was approximately 70%, which is higher than in other studies, but the mortality rate was not found to be significantly different between Candida species can differ among hospitals, and that antifungal susceptibility testing was not routinely performed in every included study patient.The limitations of this study include its single-center retrospective cohort design, the fact that the epidemiology of C. tropicalis infection. Due to the observed high resistance of C. tropicalis to fluconazole, fluconazole should not be used for empirical antifungal treatment in these patients.In candidemia patients, chronic liver disease, neutropenia, and male gender were found to be independently associated with


Oenococcus oeni strains were tested for their ability to adhere to polystyrene using mMRS and wine as culture media. Moreover, planktonic and biofilm-detached cells were investigated for their influence on malic acid degradation kinetics and aroma compound production. Three strains were able to adhere on polystyrene plates in a strain-dependent way. In particular, MALOBACT-T1 and ISO359 strains mainly grew as planktonic cells, while the ISO360 strain was found prevalent in sessile state. The strain-dependent adhesion ability was confirmed by confocal laser scanning microscopy. Planktonic and biofilm detached cells showed a different metabolism. In fact, biofilm-detached cells had a better malic acid degradation kinetic and influenced the aroma composition of resulting wines, acting on the final concentration of esters, higher alcohols, and organic acids. Oenococcus oeni in biofilm lifestyle seems to be a suitable tool to improve malolactic fermentation outcome, and to contribute to wine aroma. The industrial-scale application of this strategy should be implemented to develop novel wine styles.In this study, nine  Oenococcus oeni, Lactiplantibacillus plantarum, and Pediococcus spp.  consists in the decarboxylation of L-malic acid to L-lactic acid by lactic acid bacteria (LAB)—mainly cus spp. . It occucus spp. . The corcus spp. . O. oenir the AF . Severalompounds . Some stompounds . The capompounds . Biofilmompounds . Those pompounds .O. oeni biofilm formation. O. oeni cells organized in biofilms on oak chips were characterized by increased tolerance to wine stresses, malolactic activities, and the modification of wood volatile composition in the resulting wines. Similar data were obtained by Over the last decade, modern oenological approaches take advantage of biofilms to improve wine quality and offer new methods of technological control over industrial processes. These approaches are based on the evidence that planktonic and sessile cells show a different metabolism . Up to nO. oeni strains isolated from organic wines were studied for their adhesion ability on abiotic surfaces. Moreover, the impact of planktonic and biofilm detached cells on the MLF outcome and the aroma profile of resulting wine was studied.Thus, in this study nine O. oeni strains  were previously isolated from Montepulciano d’Abruzzo organic wines undergoing spontaneous MLF as reported by Nine Strains were grown in mMRS as previously described and then collected by centrifugation and suspended in a saline solution (8.5 g/l) to yield an initial concentration of 6 log CFU/ml. The ability to form biofilms was monitored growing the cells in flat-bottomed six-well cell culture plates  containing 5 ml of mMRS, or organic wine . Plates were incubated at 28°C under anaerobic conditions for 20 days. Uninoculated wells were used as negative controls. Media and planktonic cells were removed rinsing with saline solution. Sessile cells were collected pipetting up and down 10 times . Both seBiofilm formation in mMRS and organic wine was examined by confocal laser scanning microscopy (CLSM) using the Nikon A1-R confocal imaging system  controlled by Nikon NIS-Elements interface . Bacterial fluorescent labeling was carried out using 1 μl of Hoechst 33342 dye 10 mg/ml . All analyses were performed in triplicate.Oenococcus oeni strains were inoculated as planktonic cells or after development as biofilm (6 log CFU/ml). In particular, sessile cells were cultivated for 20 days in 20 ml of organic red wine in polystyrene plates. The 20-day-old biofilm was detached from the plate and inoculated into the wine. Similarly, planktonic cells were collected and used as inoculum. The malic acid degradation was determined according to g, the samples were diluted five-fold with 22.5 mM H2SO4 solution (mobile phase) prior to injection of 10 μl into HPLC. The aroma profile was determined as reported below.Analyses were carried out using the organic red wine previously described. Aroma compounds were determined by solid phase microextraction coupled with gas chromatography (GC-MS-SPME) using a GC-mass spectrometer Clarus SQ 8 S chromatograph/mass (GC-MS) spectrometer  as previously described . Volatilpost hoc analysis was performed with multiple t-tests. A false discovery rate correction was implemented to prevent the false-positive occurrence. A level of p < 0.05 was considered statistically significant. Principal component analysis (PCA) based on the main volatile compounds released by strains was performed using XLSTAT 2014 software . For malic acid degradation, a repeated-measure ANOVA (RM-ANOVA) was performed, together with a post hoc analysis.Data were analyzed by means of Prism 7.0 program . The data were analyzed using a two-way ANOVA, considering the media and the lifestyle as the two factors. A O. oeni were tested for their ability to adhere on polystyrene plates and only three strains—MALOBACT-T1, ISO359, ISO360—showed this capacity. The MALOBACT-T1 strain mainly developed as planktonic cells in both media, while the ISO359 strain mainly grew as planktonic cells in wine. The statistical analysis delivered a significant difference concerning the lifestyles; hence, a post hoc analysis associated with this factor was performed. The ISO360 strain showed similar values of plate counts in all conditions tested , and ISO359 and ISO360 strains showed values of 1.6 and 1.55 g/l, respectively. Detached cells had a better degradation ability. In fact, after 5 days MALOBACT-T1, ISO359, and ISO360 strains showed degradation values of 1.8, 1.45, and 1.7 g/l, respectively. After 10 days, MALOBACT-T1 consumed all the malic acid; the others showed a very low malic acid residual concentration (0.05 g/l)  .Oenococcus oeni shows several metabolic pathways and enzymes involved in volatile secondary compound production, including esters, higher alcohols, carbonyls, volatile fatty acids, and sulfur compounds  (O. oeni planktonic cells induced a reduction of organic acids.ompounds . Planktoompounds . The totompounds . A strai14 μg/l) . FinallyO. oeni lifestyle on the release of specific aroma compounds, the cells detached from biofilm were inoculated in wine as reported in Materials and Methods.To evaluate the influence of Cells detached from biofilms released a total of 47 compounds in wine . Higher O. oeni inoculated as planktonic cells and were differentiated for acetic acid, 2-phenylethyl ester, 1-butanol, 3-methyl acetate, octanoic acid, ethyl ester, and pentanoic acid, 2,2-dimethyl, methyl ester. Wines obtained with O. oeni cells detached from biofilm belonged to the II group and were well differentiated for dodecanoic acid, ethyl ester, ethyl lactate, ethyl ethanoate, phenylethyl alcohol, hexanoic acid, and ethyl ester. Obtained data suggested that O. oeni lifestyle exerts an effect in the definition of wine volatilome, suggesting that it should be possible to modulate wine characteristics using different inoculum strategies.Principal component analysis allowed 88.9% of the total variance to be explained by the first two PCs , and 71.O. oeni strains by other authors who observed the ability of this species to adhere on different materials, e.g., polystyrene, oak chips, and stainless steel surfaces  represent the potential of its exploitation to modulate MLF outcome and wine aroma development in order to develop novel wine styles. Further studies will be performed, at industrial level, to validate the role of biofilm-detached cells of O. oeni and eventually propose a new way to develop starter cultures.This study highlighted that biofilm-detached cells of The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.RT: conceptualization, investigation, writing—review and editing, and funding acquisition. GP: conceptualization, writing—original draft, writing—review and editing, and data analysis. NB, LV, and AR: methodology. CP and CZ: funding acquisition. GA: review and editing. All authors contributed to the article and approved the submitted version.CZ is employed by Orsogna winery and Carlo Perla by Dalton Biotecnologie s.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mycobacterium marseillense is a new species of the Mycobacterium avium complex. There has been only a few human infections caused by M. marseillense worldwide.M. marseillense in an immunocompetent adult in Japan. The isolate was initially identified as M. intracellulare using commercial real time polymerase chain reaction assays and later identified as M. marseillense with sequencing of the the rpoB and hsp65 regions, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS).We report a case of tenosynovitis caused by M. marseillense generating a positive result with commercial real time PCR assays targeting MAC. Human infections associated by M. marseillense might be underreported due to similarities with Mycobacterium intracellulare. To accurately identify M. marseillese, MALDI-TOF MS might provide a rapid and reliable method.This is the first case reporting on  Mycobacterium avium complex (MAC) is the most common non-tuberculosis mycobacteria (NTM) causing human infections; these include chronic pulmonary infections in adults and lymphadenitis in children. M. avium and M. intracellulare are the best-known representative species of the MAC. However, with advance in molecular analyses, multiple species have been recognized as members of the MAC, resulting in frequent emendation in the taxonomy of this species complex [Mycobacterium marseillense is among these [M. marseillense that was initially identified as M. intracellulare using commercial real time polymerase chain reaction (PCR) assays.The  complex . Althoug complex . Mycobacng these ; howeverM. intracellulare as well. However, further identification of the isolate using matrix-assisted laser desorption ionization–time of flight mass spectrometry  suggested that the isolate was M. marseillense, with a score of 2.023 indicating a probable species identification. Because of this discordance, the rpoB and hsp65 regions were sequenced [Mycobacterium marseillense (GenBank accession number CP023147.1).In March 2018, an 85-year-old man with no medical history was admitted after a 6-month history of worsening pain and swelling in the right wrist. Magnetic resonance imaging showed a large region of tenosynovitis from the distal forearm to the palm, and a biopsy of the synovial membrane was performed. Histological testing showed granulomatous inflammation. After nine weeks of culture on Ogawa medium , smooth non-pigmented colonies were observed. The colony was positive for Ziehl Neelsen stain, suggesting acid fast bacilli. Initial testing with AccuProbe  was positive for MAC, with a value of 197,546 relative light units (RLU). COBAS TaqMan MTB/MAI , another real-time PCR test, was positive for Drug susceptibility testing was performed by a broth microdilution test, BrothMIC NTM . Based on the breakpoints proposed by the Clinical and Laboratory Standards Institute , the isoM. marseillense in an immunocompetent adult. Thus far, five human infections of M. marseillense have been reported from Italy, China and the US , leading to a delay in accurate diagnosis [Commercial probes for the clinical important mycobacteria are widely used in clinical settings. Among these, AccuProbe is an FDA-approved polymerase chain reaction (PCR) test used to identify several clinically important mycobacteria, including kansasii . The idekansasii . Nonethereported , 12. To iagnosis .hsp65 sequencing to the species level [M. massiliense,  and M. gordonae  [M. massilense was not included at the time of the study but has later been added in the most recent database (MALDI Biotyper Version 6). Other studies have also reported similar results reporting a 92.0 to 97.6 % agreement between MALDI-TOF MS and either 16S rRNA, rpoB or hsp65 sequencing [MALDI-TOF MS may be of great help for mycobacterial identification in clinical microbiology laboratories. In a study of 125 isolates from 27 different NTM species, identification using MALDI-TOF MS with a cut-off score of 1.7 had a 94.4 % (118/125) agreement between 16 S rRNA and es level . The sev result) . M. massquencing , 15.M. marseillense in an immunocompetent man. Given the limited availability of sequencing in clinical settings, cases of M. marseillense infection might have been misidentified and, therefore, underreported. To understand the epidemiology of M. marseillense and its role in human infections, accurate identification is crucial, and MALDI-TOF MS might provide a rapid and reliable identification when sequencing specific regions is not readily available.In conclusion, we report on a case of tenosynovitis of the wrist by


The hunting billbug is the most dominant and damaging insect pest species of sod farms  in Georgia (USA). The larvae feed within the turfgrass stem, and roots affect turfgrass growth. Hunting billbugs are usually managed using insecticides. However, the application of insecticides to entire sod fields is not an economically and practically feasible option. Thus, an improved sampling plan for larvae and adults is warranted to improve management decisions. The current study was aimed at understanding the spatial distributions of hunting billbug larvae and adults in sod farms using geospatial techniques. The larvae and adults were sampled using soil cores and pitfall traps, respectively. After evaluating two geospatial techniques, the distribution pattern of hunting billbug larvae and adults within the sod farms was aggregated. The presence of billbugs in samples collected at 4 m apart suggests active infestation. This information will help develop integrated pest management for hunting billbug in sod farms and reduce insecticide use, benefiting growers and the environment alike.Sphenophorus venatus vestitus Chittenden (Coleoptera: Curculionidae), is an important turfgrass pest, especially in sod farms. S. venatus vestitus larvae feed on the stems and roots of turfgrass. Damaged turfgrass is loosely held together and poses a challenge for machine harvesting. Additionally, the normal growth of turfgrass is affected, especially after winter dormancy. Because S. venatus vestitus larvae are hidden inside the stems or under the soil, larval management is challenging. To improve sampling and management, the spatial distribution patterns of S. venatus vestitus larvae and adults were assessed at four sod farm sites with a history of S. venatus vestitus infestation in central Georgia (USA). The larvae were sampled by soil cores using a hole cutter, whereas adults were collected using pitfall traps for 7 d. The spatial distributions of larvae and adults was analyzed using SADIE and variograms. The SADIE and variogram analyses revealed a significant aggregation pattern for adults, whereas aggregated distributions were detected for larvae with variogram analyses. The average ranges of spatial dependence for larval and adult samples were 3.9 m and 5.4 m, respectively. Interpolated distribution maps were created to visually depict S. venatus vestitus infestation hotspots within the sod farms.The hunting billbug,  Sphenophorus venatus vestitus Chittenden (Coleoptera: Curculionidae), is a serious pest of warm-season turfgrass in the USA  and their egg parasitoid Paratelenomus saccharalis Dodd (Hymenoptera: Platygastridae) in soybean [Gossypium hirsutum L. [Oulema melanopus [L.] in wheat (Triticum aestivum L.) [S. venatus vestitus in sod farms have not been studied. Obtaining this information could help develop an effective sampling plan and improve insecticide application strategies for S. venatus vestitus control.There are many examples of how spatial distribution information can be used to understand the ecology and management of arthropod pests. A spatial distribution study of the annual bluegrass weevil fairways . Additioineyards ; alfalfaerus L.) ; Kudzu b soybean ; thrips sutum L. ; and cerivum L.) . The spaS. venatus vestitus is managed by using insecticides [S. venatus vestitus incidence in a specific field. Here, an improved sampling method for S. venatus vestitus could be beneficial and will aid management decisions. Currently, there are no sampling plans for growers that guide S. venatus vestitus management decisions. The current plans mostly depend on visual inspections around the pavements for walking adult S. venatus vestitus that are conducted early in the morning, approximately one hour after sunrise. An understanding of how S. venatus vestitus is spatially distributed within sod fields will help to develop an effective sampling strategy using available monitoring tools.On sod farms, cticides . BecauseS. venatus vestitus in Georgia sod farms. We used variograms and SADIE to characterize the spatial distribution of S. venatus vestitus adults and Sphenophorus spp. larvae. More than 98% of Sphenophorus spp. adults sampled were S.venatus vestitus, while the remaining were S. cariosus Olivier and S. inaequalis Say. Thus, the larval samples could include other Sphenophorus spp. species, and there are no morphological keys available to easily distinguish billbug larvae.Several geospatial methods, such as spatial analysis by distance indices (SADIE) and variograms, can be used to assess insect spatial distributions ,23,24,25japonica) zoysiagrass  and a ‘TifWay’ bermudagrass . In 2020, the turfgrass genotypes at two different sites were ‘Zeon’ (Z. matrella) zoysiagrass  and ‘TifTuf’ bermudagrass .Four sod fields with a history of billbug infestations in Marshallville, Georgia, USA were selected for this study. In 2019, the turfgrass genotypes in the two sod field sites were a ‘Zenith’ , fertilizer and irrigation regimes.In 2019 and 2020, d Joseph , the S. ing year . Thus, t studies ,31. ThreThe sampling plan consisted of 90 sample points in a square grid, with ~3 m between any two sample points and covered a total of 27 m × 30 m (length × width) of the field. Nine sampling points for sites 2, 3, and 4 were along the X coordinate and ten points were along the Y coordinate. For Site 1, ten sampling points were along the X coordinate, and nine points were along the Y coordinate. Larval sampling for sites 1 and 2 was initiated on 30 September and 15 October 2019, respectively, and was completed on 14 December for both sites . Larval In the laboratory, larvae were extracted from the soil core samples contained grass roots, thatch, and soil. Adult sampling for site 2 was initiated on 26 May and completed on 8 June 2020. For site 3, sampling started on 27 May and was completed on 10 June 2020. Adult sampling for site 4 began on 10 June and ended on 24 June 2020. The adults were sampled using pitfall traps that were constructed by using 11.5-cm diameter and 7.5-cm deep clear plastic containers. The containers were partially closed with Styrofoam plates to prevent rainwater from entering the traps, and ethyl glycol was added, which acted as a preservative agent for the insects. Ninety traps were deployed at each site, and the traps were monitored weekly and were kept in place for three weeks after the date of installation. The trap contents were filtered using a sieve and were transported to the laboratory for further identification. Two geospatial methods, variograms and SADIE, were used to characterize the spatial distribution patterns of billbugs within the fields.γ) can be represented byh) is the estimated semivariance for the entity of interest (z) at all points (xi), which are separated by lag distance (h), and n(h) is the number of sample pairs which are separated by lag distance h [Variograms are a commonly used method for depicting the spatial dependency of sample points. Spatial dependence is determined by developing an experimental semivariogram. Mathematically, the semivariogram (stance h .0 + C), and nugget (C0), and the values of these parameters determine the shape of the variogram. The semivariance value at which the variogram plot reaches a plateau is the sill, while the semivariance value at zero lag distance is called the nugget [γ2 value or the lowest residual sum of squares (RSS) [0/C0 + C) and nugget were used to determine the degree of aggregation [All variogram models were created using the geostatistical software GS+ . Variogram models have three parameters, range, sill  ,35. Curves (RSS) ,36,37. Aes (RSS) , and thees (RSS) ,15,25,39regation , where rregation ,41,42,43regation ,44,45,46SADIE was used to characterize billbug spatial distribution patterns and test whether the resulting distributions were statistically significant ,18. CharIa, with an associated probability, pa. Aggregated, uniform, and random distribution patterns are indicated by Ia > 1, Ia = 1, and Ia < 1, respectively [Pa < 0.025) determines whether the resultant distribution pattern is significantly different from randomness [iv with an associated p-value, Piv. In contrast, mean cluster indices that represent all units in a gap are denoted by jv with an associated p-value, Pvj. Values of iPv and jPv < 0.0025 indicate statistically significant gaps and patches, respectively. Calculations of the aggregation index and index of clustering in SADIE were carried out using SADIEShell .SADIE measures the overall aggregation based on the distance to regularity (D), which represents the minimum total distance that individuals would need to move to achieve the same number  for each sample point. The magnitude of D is assessed by a randomization test in which permutations of all observed counts among the sample points are performed . This asectively . The assndomness ,17,52. FSphenophorus spp. larvae . Based on the r2 and RSS values, the linear model fitted best for site 1 (r2 = 0.64) (r2 = 0.03) (r2 = 0.07)  out of the four sites. These results were based on the variogram model, high r site 1 A, the Ga = 0.64) B, the ex = 0.03) C and the = 0.07) D. For siS. venatus vestitus populations. Variogram analyses were conducted separately on adult data for each week and cumulatively  for all three sites. For the cumulative samples, the best-fitting variogram used the exponential model at all three sites   and 3 , whereas at site 4, spatial aggregation was not evident in the second sample  for the adult ctively) A–C. Spatd sample . The besd sample . At sited sample . For sitSphenophorus spp. or S. venatus vestitus. For Sphenophorus spp. larvae, the range values for these sites were between 3.82 and 4.11 m   . The weeng weeks .S. venatus vestitus followed spatially dependent distributions in the sod farms. The variogram results for Sphenophorus spp. larvae showed that they were spatially aggregated in 3 out of 4 sites studied. Although S. venatus vestitus mostly overwinter as adults [S. venatus vestitus were found in the sod farms in central Georgia. The larval stages sampled in fall or winter and spring are likely from the same overlapping generations because adult emergence was continuous from late winter to summer in the central Georgia sod farms [The spatial distribution of an insect is an inherited trait, but can be influenced by behavior and various environmental factors ,53. The s adults , multiplS. venatus vestitus is the dominant billbug species that causes damage (>98% of Sphenophorus spp. collected) in sod farms [S. venatus vestitus larvae and, hereafter, are referred to as S. venatus vestitus larvae. Likewise, S. venatus vestitus adults were most abundant in the fully grown sod fields [L. maculicollis, where L. maculicollis in golf courses was initially found to be aggregated at the edges of golf courses and then eventually dispersed throughout the entire course [L. maculicollis, however, overwinters in leaf litter off-site and moves into golf courses during spring every year. Thus, knowledge of the aggregated distributions of S. venatus vestitus in sod farms will help in the development of more effective IPM.A previous study showed that od farms . Thus, ad fields . They frd fields . This sue course . L. macuS. venatus vestitus distributions and 5.4 m  for the adult S. venatus vestitus distributions. Range values can be used either to create hotspot maps for site-specific management [S. venatus vestitus hotspot maps, the distance between two samples should be less than 3.9 m and 5.4 m for larvae and adults, respectively. Hotspot maps indicate those areas with high degrees of infestation and therefore, they help with information-based decision-making for pest management [S. venatus vestitus larvae are hidden in the soil, and thus, their infestations in soils can be determined if soil samples are collected using a hole cutter at 4.0 m  distances to capture larvae. Because the larval samples were mainly collected in winter and spring, further research is warranted to determine the larval distribution of S. venatus vestitus in summer and early fall. Similarly, the prevalence of S. venatus vestitus adults can be determined if they are collected by deploying pitfall traps at 5 m  distances at 7 d intervals. This information can be used for pest management decisions.Understanding spatial distributions helps to predict and manage pest populations by implementing accurate sampling plans and decision-making processes . When usnagement ,58 or tonagement . In the nagement . Howevernagement ,59. Whennagement ,26,58. SS. venatus vestitus larvae and adults showed aggregations in the variogram analyses. However, only for S. venatus vestitus adults did the data support aggregation when using SADIE analysis. This discrepancy may be due to the variations in which the spatial weights are calculated for individual sample points [Cerotoma trifurcata Forster (Coleoptera: Chrysomelidae) [Ricania shantungensis (Hemiptera: Ricaniidae) in chestnut fields [The variogram and SADIE analyses showed inconsistent results, in which both e points ,61. SADIe points ,62. As ae points . Therefoe points . A previmelidae) . Anothert fields . A previt fields ,60. Moret fields . While vt fields ,59. The t fields ,64.S. venatus vestitus in sod farms. Although most previous studies have used adult S. venatus vestitus sampling, larval sampling remains challenging [S. venatus vestitus larvae cause more economic damage than adults [S. venatus vestitus larvae is critical to developing an effective strategy for addressing this pest from a management standpoint. The current practice of larval sampling using hole cutters requires more labor and time and has still not been demonstrated to be an efficient method for determining Sphenophorus spp. larvae distributions [S. venatus vestitus larvae in the field, especially in the winter and spring months. Further research is warranted to determine the minimum number of samples per sod field to quantify S. venatus vestitus and develop thresholds for management decisions. This information can be used for spot applications or site-specific management of S. venatus vestitus larvae and S. venatus vestitus adults in sod farms and can reduce insecticide use and application costs.This is the first study that shows the spatial distributions of llenging ,5,31. Mo