Unusual Sequence Research Articles

Spatiotemporal Behavior of an Extremely Small Seismic Swarm in Pyrenean Foreland, France

ABSTRACT During the large-N MAUPASACQ passive seismic experiment in the foreland of the western Pyrenees (southwest France), an unusual swarm-type seismic sequence was serendipitously recorded in a normally quiet area. Thanks to the density of the deployment and the proximity of all events, it was possible to relocate the hypocenters with a very good relative accuracy through template matching, cross-correlation phase picks, and double-difference algorithm. The four-month seismic activity consists of more than 600 events with local magnitudes ranging between −1.4 and 2.1, clustered in an extremely small volume, and rooted at 4 km depth. The sequence can be divided in two phases of similar durations and event occurrence rates, but of different magnitude–frequency distributions. The presence of an asperity is suggested by the relative abundance of stronger events during the second phase. Fine mapping suggests a small but clear geographic offset of a few tens of meters between the events of the two phases and a very slow migration suggesting a process involving fluids. Changes in the correlation matrices and waveforms of late arrivals at a specific station are also observed, arguing either (and again) for migration of the hypocenters or for changes in the propagation medium between the two phases. The geographical coincidence with the repeated observation of hydrogen leaks on the surface, almost above the swarm, suggests a connection through channels that could carry fluids.

A corazonin G protein-coupled receptor gene in the tick Ixodes scapularis yields two splice variants, each coding for a specific corazonin receptor

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G16. CRZ-Ra turned out to be selective for I. scapularis corazonin (EC50 = 10−8 M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC50 = 4 x 10−8 M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene.

Enantioselective Total Synthesis of (+)-KB343.

A concise total synthesis of the complex guanidinium toxin KB343 is reported traversing through an unusual sequence of chemoselective transformations and strategic skeletal reorganization. The absolute configuration is confirmed through an enantioselective route, and the structures of all key intermediates and the natural product itself are unassailably confirmed through X-ray crystallographic analysis.

Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay

Actin-related protein 5 (ARP5) inhibits the differentiation of skeletal, smooth, and cardiac muscle tissues, and ARP5 expression increases or decreases according to physiological and pathological changes in the muscle differentiation status. However, the regulatory mechanisms of ARP5 expression are largely unknown. Here, we identified a novel Arp5 mRNA isoform that contains premature termination codons in alternative exon 7b and is thus targeted by nonsense-mediated mRNA decay (NMD). In mouse skeletal muscle cells, switching from the canonical Arp5 isoform, i.e., Arp5(7a), to the NMD-targeted isoform Arp5(7b) occurred during differentiation, suggesting that Arp5 expression is regulated by alternative splicing coupled to NMD (AS-NMD). We developed an original method to accurately quantify the proportion of both Arp5 isoforms and measured higher levels of Arp5(7b) in muscle and brain tissues, where ARP5 is less expressed. The 3′ splice site in Arp5 exon 7 has an unusual acceptor sequence that often leads to the skip of the authentic splice site and the use of the cryptic splice site localized 16 bases downstream. When the unusual acceptor sequence was mutated to the usual one, the Arp5(7b) isoform was barely detectable. The expression of several splicing factors involved in 3′ splice site recognition was reduced after muscle differentiation. Additionally, knockdown of splicing factors increased the levels of Arp5(7b) and decreased the expression of Arp5(7a). Furthermore, strong positive correlations were found between Arp5 expression and the levels of these splicing factors in human skeletal and cardiac muscle tissues. Thus, Arp5 expression in muscle tissues is most likely regulated by the AS-NMD pathway.

Silver-Mediated Cascade Synthesis of Functionalized 1,4-Dihydro-2H-benzo-1,3-oxazin-2-ones from Carbon Dioxide.

A conceptually novel catalytic domino approach is presented for the synthesis of highly functional 1,4-dihydro-2H-1,3-benzoxazine-2-one derivatives. Key to the chemoselectivity is a proper design of the precursor to override thermodynamically favored parasitic cyclization processes and empower the formation of the desired product through Thorpe-Ingold effects. The synthetic diversity of these CO2 -based heterocycles is further demonstrated, and the isolation of a reaction intermediate supports an unusual ring-expansion sequence from an α-alkylidene, five-membered cyclic carbonate to a six-membered cyclic carbamate by N-induced isomerization.

The unusual chemical sequences of mammalian dihydropyrimidine dehydrogenase revealed by transient-state analysis.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of the 5,6-vinylic bond of uracil and thymine with electrons from NADPH. The complexity of the enzyme belies the simplicity of the reaction catalyzed. To accomplish this chemistry DPD has two active sites that are ∼60Å apart, both of which house flavin cofactors, FAD and FMN. The FAD site interacts with NADPH, while the FMN site with pyrimidines. The distance between the flavins is spanned by four Fe4S4 centers. Though DPD has been studied for nearly 50years, it is only recently that the novel apects of its mechanism have been described. The primary reason for this is that the chemistry of DPD is not portrayed adequately by known descriptive steady-state mechanism categories. The highly chromophoric nature of the enzyme has recently been exploited in transient-state to document unexpected reaction sequences. Specifically, DPD undergoes reductive activation prior to catalytic turnover. Two electrons are taken up from NADPH and transmitted via the FAD and Fe4S4 centers to form the FAD•4(Fe4S4)•FMNH2 form of the enzyme. This form of the enzyme will only reduce pyrimidine substrates in the presence NADPH, establishing that hydride transfer to the pyrimidine precedes reductive reactivation that reinstates the active form of the enzyme. DPD is therefore the first flavoprotein dehydrogenase known to complete the oxidative half-reaction prior to the reductive half-reaction. Here we describe the methods and deduction that led to this mechanistic assignment.

1226. Two consecutive outbreaks of carbapenemase-producing enterobacteriaceae (CPE) in a pediatric hospital

Abstract Background Carbapenemase-producing enterobacteriaceae (CPE) can cause hospital outbreaks with considerable health and economic implications. Intensive infection control measures are implemented to identify CPE carriers and contain outbreaks. Methods We describe a CPE-NDM (New Delhi Metallo-beta-lactamase) hospital wide outbreak, in a tertiary care pediatric hospital, which evolved into a CPE-KPC (Klebsiella pneumoniae carbapenemase) outbreak before subsiding. Results At 10/20/21 rectal screening for CPE was performed in patients in the NICU (neonatal intensive care unit) because of known CPE carriers hospitalized there. Multiple CPE carriers were discovered, and a hospital-wide outbreak of CPE-NDM was identified. All patients who were potential contacts of CPE carriers throughout the hospital were isolated and screened twice a week, as part of intensive infection control measures. Between 10/20/21 and 12/07/21 46 patients carrying CPE-NDM were discovered. Continued screening between 12/15/21 and 04/18/22 identified 81 additional patients carrying CPE, however the carbapenemases identified in 78 of them were KPC. No known CPE-KPC carrier was hospitalized at the beginning of the KPC outbreak. Surprisingly, in 6 of the patients carrying CPE-NDM, repeat screening done months later found the carriage shifted from CPE-NDM to CPE-KPC. The results were validated and analyzed at the National Infection Control Laboratory. No significant morbidity or mortality was observed due to CPE carriage. CPE identification distribution by week and mechanism Conclusion We report an unusual sequence of two CPE outbreaks. We found no previous reports of consecutive outbreaks of CPE. Disclosures All Authors: No reported disclosures.

Unusual stacking sequence of MoS2 and WS2 vertical heterostructures in one-pot chemical vapor deposition growth

Unusual stacking sequence of MoS2 and WS2 vertical heterostructures in one-pot chemical vapor deposition growth

Phase transition hysteresis at the antiferroelectric-ferroelectric boundary in PbZr1−xTixO3

$\mathrm{Pb}{\mathrm{Zr}}_{1\ensuremath{-}x}{\mathrm{Ti}}_{x}{\mathrm{O}}_{3}$ exhibits an antiferroelectric-ferroelectric phase boundary at the composition $x\ensuremath{\sim}0.06$. Around this boundary, several questions need to be answered, such as the stabilizations of different phases with a small amount of compositional difference, the sequence of phase transitions, the coexistence of crystal structures, and potential applications. In this work, we have carried out systematic structural investigations of single crystals and ceramics with several compositions across the phase boundary. The phase diagram for $x\ensuremath{\le}0.07$ has been established. A complex phase coexistence is found near the phase boundary, which leads to an unusual transition sequence. It is confirmed that $Pbam$ and $R3c$ structures maintain a subtle balance at the phase boundary, which may be perturbed by a slight change in the concentration or external stimuli. These findings provide unique insights into understanding the antiferroelectric-ferroelectric competition and, hence, into designing alternative materials for energy storage and conversion.

Characteristics of extreme daily precipitation events over the Canadian Arctic

AbstractGiven growing interest in extreme high‐latitude weather events, we use records from nine meteorological stations and atmospheric reanalysis data to examine extreme daily precipitation events (leading, 99th and 95th percentile) over Arctic Canada. Leading events span 90 mm at Cape Dyer, along the southeast coast of Baffin Island, to 26 mm at Sachs Harbour, on the southwest coast of Banks Island. The 95th percentiles range from 20 to 30% of leading event sizes. Extreme events are most common on or near the month of climatological peak precipitation. Contrasting with Eurasian continental sites having a July precipitation peak corresponding to the seasonal peak in precipitable water, seasonal cycles in precipitation and the frequency of extremes over Arctic Canada are more varied, reflecting marine influences. At Cape Dyer and Clyde River, mean precipitation and the frequency of extremes peak in October when the atmosphere is quickly cooling, promoting strong evaporation from Baffin Bay. At all stations, leading events involved snowfall and strong winds and were associated with cyclone passages (mostly of relatively strong storms). They also involved strong vapour fluxes, sometimes associated with atmospheric rivers or their remnants. The most unusual sequence of events identified here occurred at Clyde River, where the three largest recorded precipitation events occurred in April of 1977. Obtaining first‐hand accounts of this series of events has proven elusive. Identified links between extreme events and atmospheric rivers demonstrates the need to better understand how the characteristics of such features will change in the future.

Cloning and functional characterization of two oxidosqualene cyclase genes from Siraitia grosvenorii

Oxidosqualene cyclases(OSCs), belonging to a multigene family, can convert a common precursor 2,3-oxidosqualene into various types of triterpene skeletons. In this study, primers were designed according to the analysis of Siraitia grosvenorii transcriptome data, and two OSC genes SgAS1(GenBank No. QDO67189.1) and SgAS2(GenBank No. QDO67190.1) were cloned. The open reading frame(ORF) of SgAS1 was 2 262 bp, encoding 754 amino acids, and the ORF of SgAS2 was 2 289 bp, encoding 762 amino acids. Real-time quantitative PCR results demonstrated that the two SgOSCs genes showed different expression patterns in stems, leaves, and different stages of fruits. Phylogenetic analysis showed that both SgAS1 and SgAS2 were clustered with β-amyrin synthases into a branch, but further functional characterization using yeast heterologous expression found that SgAS1 was inactive and SgAS2 could produce β-amyrin as the sole product. Multiple sequence alignments revealed that SgAS2 had a conserved MWCYCR sequence related to β-amyrin biosynthesis, while SgAS1 had an unusual LFCYTR sequence, for which the authors performed site-directed mutagenesis analysis of this sequence and found that tryptophan residue(W) was the key amino acid residue that affected the function of SgOSCs. In addition, the authors transformed the monofunctional β-amyrin synthase SgAS2 into the chassis strain GH1, which was previously modified by the research group, and increased the yield of β-amyrin to 44.05 mg·L~(-1). This study first reported the monofunctional β-amyrin synthase SgAS2 from S. grosvenorii and conducted site-directed mutagenesis and synthetic biology investigation on it, providing a valuable resource for the directed biosynthesis of triterpenoids.

Characterization of light chain c-terminal extension sequence variant in one bispecific antibody.

Protein modifications such as post-translational modifications (PTMs) and sequence variants (SVs) occur frequently during protein biosynthesis and have received great attention by biopharma industry and regulatory agencies. In this study, an aberrant peak near light chain (LC) was observed in the non-reduced capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS) electrophoretogram during cell line development of one bispecific antibody (BsAb) product, and the detected mass was about 944 Da higher than LC. The corresponding peak was then enriched by denaturing size-exclusion chromatography (SEC-HPLC) and further characterized by nrCE-SDS and peptide mapping analyses. De novo mass spectra/mass spectra (MS/MS) analysis revealed that the aberrant peak was LC related sequence variant, with the truncated C-terminal sequence “SFNR” (“GEC”deleted) linked with downstream SV40 promotor sequence “EAEAASASELFQ”. The unusual sequence was further confirmed by comparing with the direct synthetic peptide “SFNREAEAASASELFQ”. It was demonstrated by mRNA sequencing of the cell pool that the sequence variant was caused by aberrant splicing at the transcription step. The prepared product containing this extension variant maintained well-folded structure and good functional properties though the LC/Heavy chain (HC) inter-chain disulfide was not formed. Several control strategies to mitigate the risk of this LC related sequence variant were also proposed.

The influence of tropical basin interactions on the 2020–2022 double-dip La Niña

The recharge oscillator mechanism suggests that a strong El Niño event can trigger a following La Niña event that sometimes lasts for two or even three years through warm water volume preconditioning within the tropical Pacific. However, a prominent and persistent “double-dip” La Niña event appeared in the boreal winters of 2020/2021 and 2021/2022 without any significant El Niño preconditioning. Here we explore the possibility that tropical basin interactions may have initiated and helped to prolong La Niña conditions over the 2-year period 2020–2022. This period was preceded by a strong positive Indian Ocean Dipole (IOD) during the boreal fall of 2019 that gave way to basin-scale warming in the Indian Ocean in early 2020 and a notable tropical Atlantic warming in the boreal winter of 2019/2020. Later, a strong Atlantic Niño developed in the boreal summer of 2021. Using composite analyses to characterize earlier double-dip La Niñas, we argue the unusual sequence of events in 2019–2021 in the Indian and Atlantic Oceans may have energized and sustained the 2020–2022 La Niña event without any significant warm water volume preconditioning within the tropical Pacific.

Biochemical characterization of a disease-causing human osteoprotegerin variant

Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.

Dynamic regulation of Zn(II) sequestration by calgranulin C.

Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)-binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)-modulated enhancement of Zn(II) sequestration by calgranulin C. Using C13 /N15 CPMG dispersion experiments we have measured pH-dependent major and minor state populations exchanging on micro-to-millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)-bound state and can be mapped to residues located in the EF-I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano-to-microsecond timescale offer insights into the role of pH-dependent electrostatic interactions in EF-hand dynamics. Our results suggest a pH-regulated dynamic equilibrium of conformations that explore a range of "closed" and partially "open" sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non-canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.

Idiosyncratic consonant clusters: a Farsi-speaking child with protracted phonological development

ABSTRACT This study presents a nonlinear phonological analysis of speech data from a Farsi-speaking child with protracted phonological development (aged 4;8) with very low accuracy on consonants. Results revealed some common phenomena (fricatives produced as stops; dorsals and non-anterior coronals produced as anterior coronals) and some uncommon phenomena (nasals produced as oral stops; voicing and devoicing of singleton obstruents in all word positions). These phenomena interacted in word-medial clusters to create an unusual sequence of two anterior-coronal or two bilabial stops, with C1 voiced and C2 voiceless, clusters which do not occur in the basic phonology of Farsi spoken by adults. We present a non-linear constraints-based analysis of the child’s speech production and a plan for intervention targeting the child’s special difficulties.

The C-terminal head domain of Burkholderia pseudomallei BpaC has a striking hydrophilic core with an extensive solvent network.

Gram‐negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane‐proximal part of the BpaC head domain shows that the domain is exclusively made of left‐handed parallel β‐roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left‐handed parallel β‐roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent—in stark contrast to the primarily positive surface charge of the left‐handed parallel β‐roll “type” protein, YadA. We propose extending the definitions of these head domains to include the BpaC‐like head domain as a separate subtype, based on its unusual sequence, position, and charge. We speculate that the function of left‐handed parallel β‐roll structures may differ depending on their position in the structure.

The Kalaotoa Fault: A Newly Identified Fault that Generated the Mw 7.3 Flores Sea Earthquake

AbstractWe reveal the existence of a previously unknown fault that generated the Mw 7.3 Flores Sea earthquake, which occurred on 14 December 2021, approximately 100 km to the north of Flores Island, in one of the most complex tectonic settings in Indonesia. We use a double-difference method to relocate the hypocenters of the mainshock and aftershocks, determine focal mechanisms using waveform inversion, and then analyze stress changes to estimate the fault type and stress transfer. Our relocated hypocenters show that this earthquake sequence ruptured on at least three segments: the source mechanism of the mainshock exhibits dextral strike-slip motion (strike N72°W and dip 78° NE) on a west–east-trending fault that we call the Kalaotoa fault, whereas rupture of the other two segments located to the west and east of the mainshock (striking west-northwest and southeast, respectively) may have been triggered by this earthquake. The Coulomb stress change imparted by the rupture of these segments on nearby faults is investigated, with a focus on regions that experience a stress increase with few associated aftershocks. Of particular interest are stress increases on the central back-arc thrust just north of Flores and the north–south-striking Selayar fault in the northwest of our study region, both of which may be at increased risk of failure as a result of this unusual earthquake sequence.

Understanding on CRISPR/Cas9 mediated cutting-edge approaches for cancer therapeutics.

The research focus on CRISPR/Cas9 has gained substantial concentration since the discovery of ‘an unusual repeat sequence’ reported by Ishino et al. (J Bacteriol 169:5429–5433, 1987) and the journey comprises the recent Nobel Prize award (2020), conferred to Emmanuelle Charpentier and Jennifer Doudna. Cumulatively, the CRISPR has a short, compact, and most discussed success of its application in becoming one of the most versatile and paradigm shifting technologies of Biological Research. Today, the CRISPR/Cas9 genome editing system is almost ubiquitously utilized in many facets of biological research where its tremendous gene manipulation capability has been harnessed to create miracles. From 2012, the CRISPR/Cas 9 system has been showcased in almost 15,000 research articles in the PubMed database, till date. Backed by some strong molecular evidence, the CRISPR system has been utilized in a few clinical trials targeted towards various pathologies. While the area covered by CRISPR is cosmic, this review will focus mostly on the utilization of CRISPR/Cas9 technology in the field of cancer therapy.

Molecular analysis for the OprD gene among Pseudomonas aueroginosa clinical isolates obtained from hospitals in Jordan.

Pseudomonas aeruginosa has increasingly been associated with the emergence of antibiotic resistance. Antibiotic resistance among P. aeruginosa isolates is an ambiguous and complicated mechanism utilizing several enzymes and structural proteins. This study was conducted to investigate the prevalence of mutations in the chromosomal OprD gene that show resistance to carbapenems among clinical isolates of P. aeruginosa. Sixty-three clinical isolates of P. aeruginosa resistant to meropenem were collected from public hospitals in Irbid city, north of Jordan. Analysis of antimicrobial susceptibility was carried out and their susceptibility was categorized. Molecular analysis of mutations in the OprD gene was performed using restriction fragment length polymorphism (RFLP) and DNA sequencing. Molecular analysis of P. aeruginosa isolates showed 52% of the common molecular modifications among the collected isolates. These alterations could be associated and affect meropenem-susceptibility rather than imipenem. The most frequent molecular changes among the resistant isolates were the F170L substitution mutation. This was detected in 22 (35%) of the isolates with an unusual insertion sequence (IS) of 100 bp within the 590 bp DNA segment downstream of the restriction site. The divergent sequence of 10 amino acids 372(VDSSSSYAGL)383 was detected in 7 (11%) of the isolates. A significant alteration in the OprD gene in P. aeruginosa clinical isolates was found. Alterations in the OprD gene could be linked to protein permeability of the outer membrane of P. aeruginosa associated with meropenem resistance. Further investigations with a larger number of bacterial isolates are needed to validate the proposed association.