Non-repetitive Sequences Research Articles

Identification of Two Potential Gene Insertion Sites for Gene Editing on the Chicken Z/W Chromosomes.

The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.

Randomness Generation for Secure Hardware Masking – Unrolled Trivium to the Rescue

Masking is a prominent strategy to protect cryptographic implementations against side-channel analysis. Its popularity arises from the exponential security gains that can be achieved for (approximately) quadratic resource utilization. Many variants of the countermeasure tailored for different optimization goals have been proposed. The common denominator among all of them is the implicit demand for robust and high entropy randomness. Simply assuming that uniformly distributed random bits are available, without taking the cost of their generation into account, leads to a poor understanding of the efficiency vs. security tradeoff of masked implementations. This is especially relevant in case of hardware masking schemes which are known to consume large amounts of random bits per cycle due to parallelism. Currently, there seems to be no consensus on how to most efficiently derive many pseudo-random bits per clock cycle from an initial seed and with properties suitable for masked hardware implementations. In this work, we evaluate a number of building blocks for this purpose and find that hardware-oriented stream ciphers like Trivium and its reduced-security variant Bivium B outperform most competitors when implemented in an unrolled fashion. Unrolled implementations of these primitives enable the flexible generation of many bits per cycle, which is crucial for satisfying the large randomness demands of state-of-the-art masking schemes. According to our analysis, only Linear Feedback Shift Registers (LFSRs), when also unrolled, are capable of producing long non-repetitive sequences of random-looking bits at a higher rate per cycle for the same or lower cost as Trivium and Bivium B. Yet, these instances do not provide black-box security as they generate only linear outputs. We experimentally demonstrate that using multiple output bits from an LFSR in the same masked implementation can violate probing security and even lead to harmful randomness cancellations. Circumventing these problems, and enabling an independent analysis of randomness generation and masking, requires the use of cryptographically stronger primitives like stream ciphers. As a result of our studies, we provide an evidence-based estimate for the cost of securely generating n fresh random bits per cycle. Depending on the desired level of black-box security and operating frequency, this cost can be as low as 20 n to 30 n ASIC gate equivalents (GE) or 3 n to 4 n FPGA look-up tables (LUTs), where n is the number of random bits required. Our results demonstrate that the cost per bit is (sometimes significantly) lower than estimated in previous works, incentivizing parallelism whenever exploitable. This provides further motivation to potentially move low randomness usage from a primary to a secondary design goal in hardware masking research.

CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.

CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.

TS-Mal: Malware detection model using temporal and structural features learning

The cyber ecosystem is facing severe threats from malware attacks, making it imperative to detect malware to safeguard a purified Internet environment. However, current studies primarily concentrate on examining the time-based correlation between APIs for malware detection while neglecting the contextual associations derived from API categories, resulting in inadequate detection performance. In this paper, we present TS-Mal, a novel Malware detection model incorporated Temporal and Structural features learning. Particularly, TS-Mal first designs a temporal vector learning method to automatically capture the evolving representation from the non-repetitive API sequences, which can efficiently pursue the attack preferences of malware. Then TS-Mal introduces heterogeneous graphs to model the interactive relationships between APIs and presents a dense-interactive structural embedding approach to generate the fine-grained API structural representation, which is capable of utilizing API category interaction information to boost detection effectiveness. Finally, TS-Mal simultaneously integrates temporal and structural attack features to accurately identify the unknown malware, effectively defending against new malware attacks. Experimental results on real-world datasets demonstrate that our proposed TS-Mal model outperforms existing state-of-the-art methods.

The chromosome number and heterochromatin distribution pattern suggest differentiation among species of the Stylosanthes scabra-seabrana complex.

The genus Stylosanthes has economic importance in semi-arid regions and requires studies that reveal complex relationships between species involving different ploidies. The aim of this study was to cytogenetically investigate accessions of Stylosanthes identified as S. scabra, in order to properly identify the number, morphology, and pattern of distribution of heterochromatin, analyzing the karyological variability of these species. Accessions with 2n=40 and 2n=20 were identified, exhibiting semi-reticulate interphase nuclei, symmetric karyotype, varied morphology, with differences in average chromosomal size, and genome length. The analysis with the fluorochromes chromomycin (CMA) and 4',6-diamidino-2-phenylindole (DAPI) allowed the visualization of two CMA+ blocks in the subterminal region of the short arm in all accessions, and DAPI+/CMA- bands in S. scabra accessions. This suggests that not only chromosomal rearrangements, but also changes in the composition of heterochromatin, may have occurred during the speciation of this genus, and that S. scabra may be undergoing chromosomal evolution based on the observed karyological differences. In addition to the ploidy level, the distribution pattern of CMA+ heterochromatin reinforces the separation between S. scabra and S. seabrana. Thus, this genus represents an interesting group of plants for further studies on the content and quantity of repetitive and non-repetitive DNA sequences.

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma.

Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.

THU209 Small INDELs In The Androgen Receptor Gene: Phenotype Implications And Mechanisms Of Mutagenesis In Androgen Insensitivity Syndrome

Abstract Disclosure: R.L. Batista: None. R.M. Ramos: None. A.F. Afonso: None. R.J. Petroli: None. M.Y. Nishi: None. M.M. Ferrari: None. S. Domenice: None. B.B. Mendonca: None. Small indels are insertions or deletions of one or more nucleotides into the DNA sequence. Indels are highly abundant in human genomes and have contributed massively to genome evolution. However, the role of indels and their underlined mechanisms of mutagenesis in Mendelian disorders still needs to be appreciated. To explore the contribution of indels in a monogenic disorder, we analyzed all indels in the androgen receptor gene, including three novel indels, using Androgen Insensitivity Syndrome (AIS) as a model. The goal was to profile indels in AIS. To build this profile, we analyzed the indel distribution through the AR coding region, compared with genomic and exonic populational data, and analyzed their impact on the AIS phenotype and their likely mechanism of mutagenesis. We present 82 different indels in the AR gene reported in individuals with AIS. In most cases (n=78; 95.1%), the phenotype is complete AIS (CAIS). There are four AR indels reported in mild AIS. Most short indels in the AR gene among AIS are frameshift indels (n=64; 79%). Indels are distributed along the entire AR gene, with NTD domain predominance (n=45; 54.9%), followed by LTD domain (n=28; 34.1%). Most are small deletions (n=49; 59.7%), followed by small insertions (n=29; 35.3%) and complex frameshift indels (n=4; 4.8%). Most indels are short (<10 bp; n=81 = 98.8%), with an average of 2.4 nucleotides by indel. Most indels are at non-repetitive sequences with no changes in copy counting (n = 68; 82.9%). Thirteen indels were identified within tandem repeats (15.8%), whereas seven indels were at homopolymers run (8.5%). Trinucleotide regions (Poly-Q, Poly-G) are prone to indels, but indels also happen in other trinucleotide stretches (Poli-L, Poly-P). Regarding the change in copy counting, thirty-nine (47.5%) indels are CCC indels. Indel reported in MAIS is all located at tandem repeats, while all at non-repetitive sequences caused CAIS. 3.65% of AR indels are at a 6bp consensus sequence (TG A/G A/G G/T A/C). In summary, indels represent a significant portion of the variants identified in the AR gene in patients with AIS. Unlike the non-synonymous variants related to this gene, there is a genotype-phenotype correlation (indel - CAIS congruency). However, small NFS indels at small triplet runs can slightly compromise AR function leading to the mild phenotype. Homopolymer runs, and trinucleotide repeats are exonic regions at risk for indels. Slippage explains half of the cases of AR indels. These results increase the molecular understanding of the androgen receptor disruption and the underlined mechanisms of indel mutagenesis in exonic regions and their implication on phenotype. Presentation: Thursday, June 15, 2023

FibH Gene Complete Sequences (FibHome) Revealed Silkworm Pedigree.

The highly repetitive and variable fibroin heavy chain (FibH) gene can be used as a silkworm identification; however, only a few complete FibH sequences are known. In this study, we extracted and examined 264 FibH gene complete sequences (FibHome) from a high-resolution silkworm pan-genome. The average FibH lengths of the wild silkworm, local, and improved strains were 19,698 bp, 16,427 bp, and 15,795 bp, respectively. All FibH sequences had a conserved 5' and 3' terminal non-repetitive (5' and 3' TNR, 99.74% and 99.99% identity, respectively) sequence and a variable repetitive core (RC). The RCs differed greatly, but they all shared the same motif. During domestication or breeding, the FibH gene mutated with hexanucleotide (GGTGCT) as the core unit. Numerous variations existed that were not unique to wild and domesticated silkworms. However, the transcriptional factor binding sites, such as fibroin modulator-binding protein, were highly conserved and had 100% identity in the FibH gene's intron and upstream sequences. The local and improved strains with the same FibH gene were divided into four families using this gene as a marker. Family I contained a maximum of 62 strains with the optional FibH (Opti-FibH, 15,960 bp) gene. This study provides new insights into FibH variations and silkworm breeding.

Genomic Sequence of Canadian Chenopodium berlandieri: A North American Wild Relative of Quinoa.

Chenopodium berlandieri (pitseed goosefoot) is a widespread native North American plant, which was cultivated and consumed by indigenous peoples prior to the arrival of European colonists. Chenopodium berlandieri is closely related to, and freely hybridizes with the domesticated South American food crop C. quinoa. As such it is a potential source of wild germplasm for breeding with C. quinoa, for improved quinoa production in North America. The C. berlandieri genome sequence could also be a useful source of information for improving quinoa adaptation. To this end, we first optimized barcode markers in two chloroplast genes, rbcL and matK. Together these markers can distinguish C. berlandieri from the morphologically similar Eurasian invasive C. album (lamb's quarters). Second, we performed whole genome sequencing and preliminary assembly of a C. berlandieri accession collected in Manitoba, Canada. Our assembly, while fragmented, is consistent with the expected allotetraploid structure containing diploid Chenopodium sub-genomes A and B. The genome of our accession is highly homozygous, with only one variant site per 3-4000 bases in non-repetitive sequences. This is consistent with predominant self-fertilization. As previously reported for the genome of a partly domesticated Mexican accession of C. berlandieri, our genome assembly is similar to that of C. quinoa. Somewhat unexpectedly, the genome of our accession had almost as many variant sites when compared to the Mexican C. berlandieri, as compared to C. quinoa. Despite the overall similarity of our genome sequence to that of C. quinoa, there are differences in genes known to be involved in the domestication or genetics of other food crops. In one example, our genome assembly appears to lack one functional copy of the SOS1 (salt overly sensitive 1) gene. SOS1 is involved in soil salinity tolerance, and by extension may be relevant to the adaptation of C. berlandieri to the wet climate of the Canadian region where it was collected. Our genome assembly will be a useful tool for the improved cultivation of quinoa in North America.

Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells

Genome architecture and organization play critical roles in cell life. However, it remains largely unknown how genomic loci are dynamically coordinated to regulate gene expression and determine cell fate at the single cell level. We have developed an inducible system which allows Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies (SIMBA) in living cells. In SIMBA, the human heterochromatin protein 1α (HP1α) is fused to mCherry and FRB, which can be induced to form biomolecular assemblies (BAs) with FKBP-scFv, guided to specific genomic loci by a nuclease-defective Cas9 (dCas9) or a transcriptional factor (TF) carrying tandem repeats of SunTag. The induced BAs can not only enhance the imaging signals at target genomic loci using a single sgRNA, either at repetitive or non-repetitive sequences, but also recruit epigenetic modulators such as histone methyltransferase SUV39H1 to locally repress transcription. As such, SIMBA can be applied to simultaneously visualize and manipulate, in principle, any genomic locus with controllable timing in living cells.

CRISPR FISHer enables high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification

The dynamic three-dimensional structures of chromatin and extrachromosomal DNA molecules regulate fundamental cellular processes and beyond. However, the visualization of specific DNA sequences in live cells, especially nonrepetitive sequences accounting for most of the genome, is still vastly challenging. Here, we introduce a robust CRISPR-mediated fluorescence in situ hybridization amplifier (CRISPR FISHer) system, which exploits engineered sgRNA and protein trimerization domain-mediated, phase separation-based exponential assembly of fluorescent proteins in the CRISPR-targeting locus, conferring enhancements in both local brightness and signal-to-background ratio and thus achieving single sgRNA-directed visualization of native nonrepetitive DNA loci in live cells. In one application, by labeling and tracking the broken ends of chromosomal fragments, CRISPR FISHer enables real-time visualization of the entire process of chromosome breakage, separation, and subsequent intra- or inter-chromosomal ends rejoining in a single live cell. Furthermore, CRISPR FISHer allows the movement of small extrachromosomal circular DNAs (eccDNAs) and invading DNAs to be recorded, revealing substantial differences in dynamic behaviors between chromosomal and extrachromosomal loci. With the potential to track any specified self or non-self DNA sequences, CRISPR FISHer dramatically broadens the scope of live-cell imaging in biological events and for biomedical diagnoses.

CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus

Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.

Fractional meanings of nonrepetitiveness

A sequence S is called r-nonrepetitive if no r sequentially adjacent blocks in S are identical. By the classic results of Thue from the beginning of the 20th century, we know that there exist arbitrarily long binary 3-nonrepetitive sequences and ternary 2-nonrepetitive sequences. This discovery stimulated over the years intensive research leading to various generalizations and many exciting problems and results in combinatorics on words.In this paper, we study two fractional versions of nonrepetitive sequences. In the first one, we demand that all subsequences of a sequence S, with gaps bounded by a fixed integer j≥1, are r-nonrepetitive. (This variant emerged from studying nonrepetitive colorings of the Euclidean plane.) Let πj(r) denote the least size of an alphabet guaranteeing existence of arbitrarily long such sequences. We prove that ⌈jr−1⌉+1≤πj(r)≤2⌈jr−1⌉+1, for all r≥3 and j≥1. We also consider a more general situation with the gap bound j being a real number, and apply this to nonrepetitive coloring of the plane.The second variant allows for using a “fractional” alphabet, analogously as for the fractional coloring of graphs. More specifically, we look for sequences of b-element subsets B1,B2,… of an a-element alphabet, with the ratio a/b as small as possible, such that every member of the Cartesian product B1×B2×⋯ is r-nonrepetitive. By using the entropy compression argument, we prove that the corresponding parameter πf(r)=inf⁡ab can be arbitrarily close to 1 for sufficiently large r.

HYDROSTATIC GENERATOR OF NON-PERIODIC PRESSURE IMPULSES FOR TESTING TECHNICAL PRODUCTS

The article reviews the possibility of using the hydrostatic generators of oscillators with nonlinear power elements (elastic shells) as part of the structures of various test benches in the field of mechanical tests for vibration strength and vibration resistance, as well as when testing with external and internal pressure. The hydrostatic generator of non-periodic pressure impulses for engineering products testing is a new patented technical solution in the field of testing machines. The ratios of the angular velocities of the shafts, which provides the movement of the operating fluid into the actuator of the test bench in the form of a non-periodic function and with impulses varying in amplitude in a random manner were obtained. It makes a continuous and non-repetitive sequence of pressure impulses and corresponds to the operation of the tested objects in real conditions. The random nature of the amplitudes settings is provided by signals controlled from electric generators through controlled hydraulic valves. The development of a hydrostatic generator with a power element in the form of an elastic shell is represented by a hydromechanical diagram of a generator of non-periodic pressure impulses with corresponding kinematic and dynamic dependencies. In addition, there were developed three hydro-mechanical systems with the use of a new hydrostatic generator for engineering products testing both for mechanical random vibration and for testing the external and internal pressure of engineering objects that operate in such conditions. The result of the research is the expansion of the range of systems and methods for the test processes implementation under random non-periodic force effects on various engineering objects.

Development of an enhanced scatter search algorithm using discrete chaotic Arnold’s cat map

Solving optimization problems is an ever-growing subject with an enormous number of algorithms. Examples of such algorithms are Scatter Search (SS) and genetic algorithms. Modifying and improving of algorithms can be done by adding diversity and guidance to them. Chaotic maps are quite sensitive to the initial point, which means even a very slight change in the value of the initial point would result in a dramatic change of the sequence produced by the chaotic map Arnold's Cat Map. Arnold's Cat Map is a chaotic map technique that provides long non-repetitive random-like sequences. Chaotic maps play an important role in improving evolutionary optimization algorithms and meta-heuristics by avoiding local optima and speeding up the convergence. This paper proposes an implementation of the scatter search algorithm with travelling salesman as a case study, then implements and compares the developed hyper Scatter Arnold's Cat Map Search (SACMS) method against the traditional Scatter Search Algorithm. SACMS is a hyper Scatter Search Algorithm with Arnold's Cat Map Chaotic Algorithm. Scatter Arnold's Cat Map Search shows promising results by decreasing the number of iterations required by the Scatter Search Algorithm to get an optimal solution(s). Travelling Salesman Problem, which is a popular and well-known optimization example, is implemented in this paper to demonstrate the results of the modified algorithm Scatter Arnold's Cat Map Search (SACMS). Implementation of both algorithms is done with the same parameters: population size, number of cities, maximum number of iterations, reference set size, etc. The results show improvement by the modified algorithm in terms of the number of iterations required by SS with an iteration reduction of 10–46 % and improvements in time to obtain solutions with 65 % time reduction

Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats.

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

Diagnostic application of sensitive and specific phage-exposed epitopes for visceral leishmaniasis and human immunodeficiency virus coinfection.

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.

The Curious Case of Nonrepetitive Centromeric DNA Sequences in Candida auris and Related Species

ABSTRACT2009 saw the first description of Candida auris, a yeast pathogen of humans. C. auris has since grown into a global problem in intensive care settings, where it causes systemic infections in patients with underlying health issues. Recent whole-genome sequencing has discerned five C. auris clades with distinct phenotypic features which display genomic divergence on a DNA sequence and a chromosome structure level. In the absence of sexual reproduction in C. auris, the mechanism(s) behind the rapid genomic evolution of this emerging killer yeast has remained obscure. Yet, one important bit of information about chromosome organization was missing, the identification of the centromeres. In a recent study, Sanyal and coworkers (A. Narayanan, R. N. Vadnala, P. Ganguly, P. Selvakumar, et al., mBio 12:e00905-21, 2021, https://doi.org/10.1128/mBio.00905-21) filled this knowledge gap by mapping the centromeres in C. auris and its close relatives. This represents a major advance in the chromosome biology of the Candida/Clavispora clade.

First mitogenome of subfamily Langiinae (Lepidoptera: Sphingidae) with its phylogenetic implications

To date, a relatively complete classification of Sphingidae (Lepidoptera) has been generated, but the phylogeny of the family remains need to be fully resolved. Some phylogenetic relationships within Sphingidae still remains uncertain, especially the taxonomic status of the subfamily Langiinae and its sole included genus and species, Langia zenzeroides. To begin to address this problem, we generated nine new complete mitochondrial genomes, including that of Langia, and together with that of Theretra oldenlandiae from our previous study and 25 other Sphingidae mitogenomes downloaded from GenBank, analyzed the phylogenetic relationships of Sphingidae and investigated the mitogenomic differences among members of the Langiinae, Sphinginae, Smerinthinae and Macroglossinae. The mitogenomes of Sphingidae varied from 14995bp to 15669bp in length. The gene order of all newly sequenced mitogenomes was identical, containing 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and the A+T-rich region. Nucleotide composition was A+T biased, and all the protein-coding genes exhibited a positive AT-skew, which was reflected in the nucleotide composition, codon, and amino acid usage. The A+T-rich region was comprised of nonrepetitive sequences, which contained regulatory elements related to the control of replication and transcription. We analyzed concatenated gene sequences, with third codon positions of protein coding genes and rRNAs excluded, using Maximum Likelihood and Bayesian Inference techniques. All four currently recognized subfamilies were recovered as monophyletic but in contrast to the most recent studies, our preferred tree placed Langiinae as the first subfamily to diverge within Sphingidae rather as sister to Smerinthinae+Sphinginae. Our results also support the removal of the genus Barbourion from the smerinthine tribe Ambulycini to an unresolved position in "Smerinthinae incertae sedis".