Alternative Primer Research Articles

Examining focus and alternative priming: Effects of grammatical role and breadth of the alternative set

Recent work has shown that contrastive accenting plays a crucial role in discourse processing, causing listeners to activate alternatives to focused words and/or suppress non-contrastive semantic associates. However, key theoretical questions remain, relating to how lexical activation, sentence and discourse processing interact. These include the breadth of the alternative set, which could span from a small contextually-relevant set to a large, ‘permissive’ one; and whether these processes are best characterised as activation or suppression mechanisms. There is also little research on whether activation of alternatives differs by the grammatical role of the prime, despite differences in the focus-related properties of subjects versus objects. We present two cross-modal lexical decision experiments showing activation of non-contrastive associates is suppressed with contrastive focus, consistent with a suppression mechanism, at least for objects. Alternatives both semantically related, and unrelated, to the prime, were primed, consistent with a broad, ‘permissive’, alternative set. There were crucial differences in priming patterns for subjects versus objects. The study makes important contributions to our theoretical understanding of the role of focus in discourse processing.

Rapid and Ultrasensitive Detection of H. aduncum via the RPA-CRISPR/Cas12a Platform.

Hysterothylacium aduncum is one of six pathogens responsible for human anisakiasis. Infection with H. aduncum can cause acute abdominal symptoms and allergic reactions and is prone to misdiagnosis in clinical practice. This study aims to enhance the efficiency and accuracy of detecting H. aduncum in food ingredients. We targeted the internal transcribed spacer 1 (ITS 1) regions of Anisakis to develop a visual screening method for detecting H. aduncum using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system. By comparing the ITS 1 region sequences of eight nematode species, we designed specific primers and CRISPR RNA (crRNA). The specificity of RPA primers was screened and evaluated, and the CRISPR system was optimized. We assessed its specificity and sensitivity and performed testing on commercial samples. The results indicated that the alternative primer ADU 1 was the most effective. The final optimized concentrations were 250 nM for Cas12a, 500 nM for crRNA, and 500 nM for ssDNA. The complete test procedure was achievable within 45 min at 37 °C, with a limit of detection (LOD) of 1.27 pg/μL. The amplified product could be directly observed using a fluorescence microscope or ultraviolet lamp. Detection results for 15 Anisakis samples were entirely consistent with those obtained via Sanger sequencing, demonstrating the higher efficacy of this method for detecting and identifying H. aduncum. This visual detection method, characterized by simple operation, visual results, high sensitivity, and specificity, meets the requirements for food safety testing and enhances monitoring efficiency.

Evaluation of cytokine release syndrome (CRS) in patients with relapsed or refractory multiple myeloma (RRMM) receiving step-up priming doses and longer dosing intervals of elranatamab: MagnetisMM-9.

7522 Background: Elranatamab (ELRA) is a humanized BCMA-CD3 bispecific antibody. In the phase 2 registrational MagnetisMM-3 (MM-3) trial, SC ELRA was given as 2 step-up priming doses (12 mg on C1D1 and 32 mg on C1D4) followed by 76 mg QW in patients (pts) with RRMM. Overall, 56.3% of pts had CRS (grade 2, 14.3%; no grade ≥3). Most events occurred after doses 1 (44.5%), 2 (20.2%), and 3 (5.9%); 0.8% (1 pt) had CRS with doses 4+. Recurrent CRS (>1 event) occurred in 15.1% of pts (Lesokhin Nat Med 2023). Methods: MagnetisMM-9 (MM-9; NCT05014412) is a phase 1/2, open-label, nonrandomized study of ELRA examining an alternative 2-dose step-up priming regimen (4 and 20 mg on C1D1 and C1D4, respectively). Eligible pts had RRMM and were refractory to ≥1 IMiD, ≥1 PI, and ≥1 anti-CD38 antibody. After the priming doses, ELRA 76 mg was given QW for 6 cycles (Part 1) or for 1 cycle followed by 116 or 152 mg Q2W for 5 cycles (Part 2A). The RP2D from Part 2A (152 mg) was evaluated in Part 2B (dose expansion). The rate of grade ≥2 CRS per ASTCT criteria during C1 is the primary endpoint for both parts. Secondary endpoints include evaluation of AEs and PK. Here, we report the overall safety and CRS profile associated with the 4/20 mg priming regimen. Results: For 85 treated pts, median age was 64.0 y; 49.4% were male; 23.5% had EMD; 31.8% had high-risk cytogenetics. Pts had a median of 5.0 (range, 1-12) prior LOTs; 85.9% had triple-class refractory disease. After a median follow-up of 7.4 mo, the most common (>50%) AEs were CRS (63.5%; grade ≥2, 15.3%) and neutropenia (54.1%; all grade ≥2). ICANS occurred in 4.7% of pts (all grade ≤2). The grade ≥2 CRS rate in C1 was 14.1% (90% CI, 8.4-21.9). CRS rates after the first 3 doses are in the the table. For doses 4+, any grade (grade ≥2) CRS was observed in 10.6% (3.5%) of pts overall, 12.1% (3.0%) of pts continuing to receive 76 mg (n=33), 25.0% (0%) of pts receiving 116 mg (n=12), and 5.0% (5.0%) of pts receiving 152 mg (RP2D; n=40). Overall, recurrent CRS was observed in 20.0% of pts. The geometric means (CV%) of free ELRA concentrations 24 h (Cmax-24h) after step-up doses 1 and 2 were 85.64 (48%) and 242.8 (55%). Conclusions: The 4/20 mg step-up priming regimen and alternative dosing schedules resulted in similar safety and incidence of overall and grade ≥2 CRS events vs the regimen used in MM-3 (12/32 mg), with no new safety signals identified. However, the CRS profile in this study differed, with more CRS after doses 2, 3, and 4+ and a higher prevalence of recurrent CRS. The Cmax-24h of free ELRA (CV%) after the priming doses were lower than those in MM-3 (107.4 [47%] and 405.1 [72%], respectively). Thus, the priming regimen used in MM-3 remains the optimal regimen for mitigating CRS. Future analyses of ongoing studies will be used to confirm these results. Clinical trial information: NCT05014412 . [Table: see text]

Low-Cost Arduino Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Sensitive Nucleic Acid Detection.

This work presents a low-cost transcription loop-mediated isothermal amplification (RT-LAMP) instrument for nucleic acid detection, employing an Arduino Nano microcontroller. The cooling system includes customized printed circuit boards (PCBs) that serve as electrical resistors and incorporate fans. An aluminum block is designed to accommodate eight vials. The system also includes two PCB heaters-one for sample heating and the other for vial lid heating to prevent condensation. The color detection system comprises a TCS3200 color 8-sensor array coupled to one side of the aluminum heater body and a white 8-LED array coupled to the other side, controlled by two Multiplexer/Demultiplexer devices. LED light passes through the sample, reaching the color sensor and conveying color information crucial for detection. The top board is maintained at 110 ± 2 °C, while the bottom board is held at 65 ± 0.5 °C throughout the RT-LAMP assay. Validation tests successfully demonstrated the efficacy of the colorimetric RT-LAMP reactions using SARS-CoV-2 RNA amplification as a sample viability test, achieving 100% sensitivity and 97.3% specificity with 66 clinical samples. Our instrument offers a cost-effective (USD 100) solution with automated result interpretation and superior sensitivity compared to visual inspection. While the prototype was tested with SARS-CoV-2 RNA samples, its versatility extends to detecting other pathogens using alternative primers, showcasing its potential for broader applications in biosensing.

Review of the state of art of Li-based inhibitors and coating technology for the corrosion protection of aluminium alloys

The quest for novel alternatives to hexavalent-chromium-based corrosion inhibitors is of utmost significance and urgency. Strict international health and safety regulations, due to growing concerns regarding the impact of hexavalent chromium on human health and the environment, have pushed the commercial introduction of many alternative inhibitor types, but the implementation of alternative active protective primers for structural parts in the aerospace industry is still pending. This endeavour has proven to be remarkably challenging, as the potential replacement coating types must meet numerous functional requirements encompassing cost-effectiveness and exceptional corrosion protection for intrinsically corrosion susceptible aerospace aluminium alloys. In recent years, considerable attention has been drawn to lithium salts as environmentally friendly corrosion inhibitors forming the basis for a novel active protective coating technology. The involvement of lithium ions has been shown to play a pivotal role in the conversion process of aluminium alloy surfaces by stabilizing the reaction products, thereby facilitating the gradual development of a protective layer with a multi-layered configuration, which exhibits considerable variability in morphology, depending on local chemical and electrochemical conditions. The versatility of the lithium-based corrosion protection extends to their application as corrosion inhibiting pigments in organic coatings or as a pre-treatment, directly forming conversion layers, thereby enhancing their practical implementation. However, previous chromate replacement reviews only introduced the promising outcomes provided by the lithium technology, omitting key details of its development and formation mechanism. This paper critically reviews and summarizes the studies conducted to date on lithium-based inhibitor technologies for the corrosion protection of aluminium alloys as well as topics to be investigated in the future.

Implicature priming, salience, and context adaptation

Recent experimental research has observed two kinds of priming effects on quantity implicatures. One is the Strong–Weak contrast, where more quantity implicatures are observed after prime trials forcing interpretations with quantity implicatures (‘Strong primes’) than after prime trials forcing interpretations without quantity implicatures (‘Weak primes’). The other effect is the Alternative-Weak contrast, where prime trials mentioning alternative expressions (‘Alternative primes’) similarly lead to more quantity implicatures. It has been claimed that both of these effects should be understood in terms of increased salience of alternative expressions used to compute quantity implicatures. We present experimental evidence that speaks against this hypothesis. With the help of novel baseline conditions, which were absent in previous studies on implicature priming, we observe that the results in the priming paradigm commonly used in the literature are inverse preference effects in the sense that robust priming effects are observed towards interpretations that are normally unexpected, and depending on the baseline expectation, each of the three prime types mentioned above may have priming effects. We furthermore investigated different types of alternative priming for so-called ad hoc implicatures and found that for these implicatures, presenting an alternative expression in a simple sentence does not have a priming effect on the implicature of a similarly simple sentence, but presenting it in a more complex conjunctive construction does. Our results also show that conjunctions of similar but irrelevant expressions have a similarly robust priming effect and that conjunctive sentences with two conjuncts do not give rise to priming effects on the interpretation of sentences of the same syntactic complexity, but those with three conjuncts do. To make sense of these observations, we propose that what crucially matters for priming implicatures is incremental change in one’s probabilistic expectations about the current conversational context brought about by a process we call context adaptation.

Improved influenza A whole-genome sequencing protocol.

Influenza A virus poses significant public health challenges due to its high mutation rate and zoonotic potential. Whole-genome sequencing (WGS) is crucial for monitoring and characterizing these viruses. Oxford Nanopore Technologies (ONT) and Illumina next-generation sequencing platforms are commonly used, with ONT being advantageous for its long-read capabilities, portability, and unique ability to access raw data in real-time during sequencing, making it suitable for rapid outbreak responses. This study optimizes the ONT Ligation Sequencing Influenza A Whole Genome protocol by refining RT-PCR kits, primers, and purification methods, and evaluating automation for high-throughput processing. The alternative RT-PCR kits, combined with alternative primers, significantly improved read depth coverage and reduced short, untargeted reads compared to the original ONT protocol. The improvement was particularly evident in the minimum read depth coverage of polymerase segments, which often face challenges with achieving uniform coverage, displaying higher coverage at the 5' and 3' termini, and lower coverage in the central regions. This optimized protocol for targeted influenza A WGS not only enhances sequencing quality and efficiency, but is applicable to all NGS platforms, making it highly valuable for studying influenza adaptation and improving surveillance. Additionally, this protocol can be further refined and adapted for the sequencing of other pathogens, broadening its utility in various pathogen monitoring and response efforts.

Hydropriming improves seed germination in horned melon (Cucumis metuliferus E. Mey. Ex Naudin) landraces

The global popularity of horned melon highlights the need of sound crop establishment processes, since uniform and quick seed germination has a substantial influence on crop yield and quality. Using a Completely Randomised Design (CRD) with a factorial structure, the effects of hydro priming durations (0, 8, 16, 24, 32, and 40 h) on seeds of two horned melon landrace (L1&L2) were investigated. Hydro priming had significant (P>0.05) influence on several germination indices (germination percentage, relativized percentage, mean germination rate, germination index, synchronization, duration to 50 % germination, mean daily germination, peak value, and germination value). Among the germination indicators tested, landrace (L2) exhibited a considerably stronger germination response than L1. The findings also reveal a curvilinear relationship between priming length and germination percentage, with 24 hours being greatest proportionately. The mean germination rate, germination index, mean daily germination, peak value of germination, and germination value all rose as the hydro priming duration increased. Primed seeds germinated more quickly than unprimed ones. With increasing priming duration, the synchronization index fell linearly. The interaction between landraces and hydro priming was not significant (P>0.05) for final seed germinated, relativized germination, and synchronization indices, but it had a significant impact on all other indices. Because the efficacy of hydro priming tends to lessen with extended soaking, we recommend using it for a maximum of 32 hours. Future study should concentrate on alternative priming chemicals and the applicability of various priming solutions for various agroecosystems

Экологические аспекты инициирующих взрывчатых веществ и перспективы их совершенствования

The main primers in the mining industry are blasting caps with highly toxic energy-intensive priming materials based on lead compounds. Unfortunately, behind the detailed complexity of modern approaches to calculate the environmental damage, it is rather difficult to assess the specific indicators of lead emissions from the blasting caps, therefore, an alternative option is presented, which allows to demonstrate a more explicit equivalent of the negative consequences. Analysis of statistical data on production and consumption of lead-containing primers indicates their significant contribution to the total and annually increasing pollution of the environment with lead, emphasizing the need to seek alternative energy-intensive priming materials. The current stage of global engineering and technological development has witnessed alternative primers already introduced into production and use, but still somewhat controversial in terms of some performance specifications and manufacturing technologies, which are utilizing new compounds that do not contain heavy metals. They are also based on fundamentally new methods of blast initiation in energy-intensive materials, in particular, using the effects of radiation from optical quantum generators (lasers). The combination of the presented environmental factors and scientific and technological achievements point to the need to intensify domestic research and development to replace highly toxic primers.

Genus-specific Primer Design for Molecular Identification of Rhodobacter sp. Origin of the 16S rRNA Gene

Rhodobacter is one of the purple phototropic bacteria widely used in aquaculture activity. It is particularly difficult in identifying and distinguishing Rhodobacter from other non-sulfur purple bacteria. This study aimed to evaluate primer pairs designed based on the 16S rRNA gene from Rhodobacter as an effective and efficient molecular identification that can be performed in the laboratory using conventional PCR techniques. The 16S rRNA sequences from Rhodobacter and other bacteria were aligned and analyzed for the high-variation region to capture the desired target amplicon. Forward and reverse primer pairs were designed based on the appropriate target amplicon. The primer pairs were also assessed for their feasibility based on ten assessment criteria for the quality of the PCR primers. The PCR optimization results obtained showed a specific band with a size of 548 bp according to the amplicon target size in the Rhodobacter samples tested and did not show any cross-reaction with other comparator bacteria, and no dimers or other non-specific bands were found. Based on the results obtained, it can be concluded that the primer pairs designed in terms of quality and specificity are good and can be used as alternative specific primers for the genus Rhodobacter sp.

Reverse transcriptases prime DNA synthesis.

The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.

Role of NPR1 in Systemic Acquired Stomatal Immunity.

Stomatal immunity is the primary gate of the plant pathogen defense system. Non-expressor of Pathogenesis Related 1 (NPR1) is the salicylic acid (SA) receptor, which is critical for stomatal defense. SA induces stomatal closure, but the specific role of NPR1 in guard cells and its contribution to systemic acquired resistance (SAR) remain largely unknown. In this study, we compared the response to pathogen attack in wild-type Arabidopsis and the npr1-1 knockout mutant in terms of stomatal movement and proteomic changes. We found that NPR1 does not regulate stomatal density, but the npr1-1 mutant failed to close stomata when under pathogen attack, resulting in more pathogens entering the leaves. Moreover, the ROS levels in the npr1-1 mutant were higher than in the wild type, and several proteins involved in carbon fixation, oxidative phosphorylation, glycolysis, and glutathione metabolism were differentially changed in abundance. Our findings suggest that mobile SAR signals alter stomatal immune response possibly by initiating ROS burst, and the npr1-1 mutant has an alternative priming effect through translational regulation.

Development of test systems for the analysis of polymorphism of the TNFAIP3 and CDS1 genes associated with the fat thickness in pigs

Relevance. In pig husbandry, one of the main issue in selection and breeding work is the efficiency of increasing the yield of quality products. The development of molecular genetic research methods using chips of various densities and subsequent genome-wide association studies made it possible to identify a large number of new genes potentially associated with selectively significant traits. Some of these potential genes are the TNFα-induced protein 3 (TNFAIP3) and CDP-diacylglycerol synthase 1 (CDS1) genes. The results of domestic and foreign studies show that these genes are associated with the regulation of the process of catabolism of cellular proteins and differentiation of fat cells.Methods. For further investigation, two polymorphisms were selected that showed a reliable association with the signs: the thickness of the fat over the 6–7 thoracic vertebrae and the thickness of the fat over the 10–12 thoracic vertebrae — in the TNFAIP3 genes (SSC1, rs81351586, A/G) and CDS1 (SSC8, rs331818788, C/A). Polymorphism was determined by real-time PCR. The selection of oligonucleotide probes and primers was carried out based on the localization of the mutation using the BLAST online resource. To test the information content of the developed RT-PCR test systems, alternative primer pairs were selected for RFLP analysis. DNA samples from 50 Large White pigs were used as genetic material.Results. The developed test systems for potential marker genes of productivity TNFAIP3 and CDS1 made it possible to clearly determine the genotypes of animals in the PCR-RT format. Both studied loci were found to be polymorphic. The developed test system can be used for genotyping a large number of animals and selecting animals with certain genotypes.

Diversity, distribution, and methodological considerations of haemosporidian infections among Galliformes in Alaska.

Using samples spanning 10-degrees of latitude in Alaska, we provide the first comparative assessment of avian haemosporidia distribution of Arctic Alaska with subarctic host populations for four species of grouse and three species of ptarmigan (Galliformes). We found a high overall prevalence for at least one haemospordian genus (88%; N=351/400), with spruce grouse (Canachites canadensis) showing the highest prevalence (100%; N=54/54). Haemoproteus and Plasmodium lineages were only observed within grouse, while Leucocytozoon species were found within both grouse and ptarmigan. Further, different Leucocytozoon lineages were obtained from blood and tissue samples from the same individual, potentially due to the differential timing and duration of blood and tissue stages. Using different primer sets, we were able to identify different Leucocytozoon lineages within 55% (N=44/80) of sequenced individuals, thereby detecting coinfections that may have otherwise gone undetected. The commonly used Haemoproteus/Plasmodium primers amplified Leucocytozoon for 90% (N=103/115) of the products sequenced, highlighting the potential value of alternate primers to identify intra-genus coinfections and the importance of obtaining sequence information rather than relying solely on PCR amplification to assess parasite diversity. Overall, this dataset provides baseline information on parasite lineage distributions to assess the range expansion associated with climate change into Arctic regions and underscores methodological considerations for future studies.

Effects of ascorbic acid addition on the oxidative stress response of Oryza sativa L. plants to As(V) exposure

Accumulation of noxious elements in the edible part of crops and its impact on food safety is of increasing concern. Rice is one of the major staple food crops worldwide, including arsenic (As)-polluted areas, in which dietary As exposure is becoming a widespread health threat. Plant chemical priming has been shown to be an effective strategy to enhance tolerance to environmental stresses, including metal(loid) exposure. The priming effect of ascorbic acid (AsA) was assessed in rice seedlings exposed to As(V) in a hydroponics experiment. AsA treatment (co-addition to the growing media concomitantly (t0) or 24 h in advance (t24)) prevented an excessive accumulation of As in the roots (that decreased ∼ 60%) and stimulated the activities of photosynthetic and antioxidant attributes (∼1.2-fold) in the aerial part of the plants. The increase in proline levels in both shoots (∼2.1-fold) and roots (∼2.4-fold) was found to be the most sensitive stress parameter, and was able to reflect the AsA-induced reduction of As toxic effects (concentrations back to Control levels, both simultaneously added or added as a pretreatment) in the aerial part of the plants. However, the phytotoxic effects related to As exposure were not fully prevented by priming with AsA, and further research is needed to find alternative priming approaches.

PB2032: MAGNETISMM-9: AN OPEN-LABEL, MULTICENTER, NON-RANDOMIZED PHASE 1/2 STUDY OF ELRANATAMAB IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

Background: Elranatamab (PF-06863135) is a humanized bispecific antibody that targets both B cell maturation antigen (BCMA) on multiple myeloma (MM) cells and CD3 on T cells resulting in T cell- mediated cytotoxicity. In preclinical studies, elranatamab has demonstrated anti-myeloma activity and delayed disease progression. In the ongoing phase 1 MagnetisMM-1 study, elranatamab has demonstrated clinical efficacy and a manageable safety profile in patients with relapsed/refractory MM (Bahlis et al, J Clin Oncol 2021). Elranatamab maximum tolerated dose has not yet been reached. Subcutaneous elranatamab at the recommended dose of 76 mg once weekly on a 28-day cycle with a 2 step-up priming dose regimen administered during the first week is currently being evaluated in a phase 2 study, MagnetisMM-3 (Lesokhin et al, Blood, 2021). Aims: To evaluate an alternative priming regimen to further mitigate cytokine release syndrome incidence and severity, and to evaluate subcutaneous elranatamab every 2 weeks and every 4 weeks in patients with relapsed/refractory MM. Methods: MagnetisMM-9 (start date: October 2021; estimated completion date: April 2025) is an open-label, multicenter, non-randomized phase 1/2 study. In part 1 of the study, patients will receive premedications and 2 step-up priming doses on cycle 1 day 1 (4 mg) and day 4 (20 mg), followed by elranatamab 76 mg once weekly for 6 cycles, and a dose every 2 weeks thereafter if partial response or better is achieved and maintained. In part 2, higher doses of elranatamab (>76 mg) at increased intervals between doses will be evaluated. The primary endpoint is grade ≥2 cytokine release syndrome rate during cycle 1. Secondary endpoints include dose-limiting toxicities (part 2 only), objective response rate, duration of response, progression-free survival, overall survival, minimal residual disease negativity rate, safety, and pharmacokinetics. Eligible patients should be 18 years of age or older with a MM diagnosis and measurable disease according to International Myeloma Working Group criteria, refractory to at least one proteasome inhibitor, one immunomodulatory drug, and one anti-CD38 antibody, relapsed/refractory to their last treatment regimen, and have an Eastern Cooperative Oncology Group performance status ≤1. Key exclusion criteria are smoldering MM, active plasma cell leukemia, amyloidosis, POEMS syndrome, stem cell transplant within 12 weeks of enrollment, active, uncontrolled bacterial, fungal, or viral infections, or any other active malignancy within 3 years prior to enrollment (except for adequately treated basal cell or squamous cell skin cancer, or carcinoma in situ), or previous treatment with an anti-BCMA bispecific antibody. The study is open at centers in the USA, UK, Japan, and Taiwan. ClinicalTrials.gov ID: NCT05014412. Results: Results not available (trial-in-progress) Summary/Conclusion: Conclusion not available (trial-in-progress)

MagnetisMM-9: An open-label, multicenter, non-randomized phase 1/2 study of elranatamab in patients with relapsed/refractory multiple myeloma.

TPS8068 Background: Elranatamab (PF-06863135) is a humanized bispecific antibody that targets both B cell maturation antigen (BCMA) on multiple myeloma (MM) cells and CD3 on T cells resulting in T cell- mediated cytotoxicity. In preclinical studies, elranatamab has demonstrated anti-myeloma activity and delayed disease progression. In the ongoing phase 1 MagnetisMM-1 study, elranatamab has demonstrated clinical efficacy and a manageable safety profile in patients with relapsed/refractory MM (Bahlis et al, J Clin Oncol 2021). Elranatamab maximum tolerated dose has not yet been reached. Subcutaneous (SC) elranatamab at the recommended dose of 76 mg QW on a 28-d cycle with a 2 step-up priming dose regimen administered during the first week is currently being evaluated in a phase 2 study, MagnetisMM-3 (Lesokhin et al, Blood, 2021). Methods: MagnetisMM-9 (start date: Oct 2021; estimated completion date: Apr 2025) is an open-label, multicenter, non-randomized phase 1/2 study evaluating an alternative priming regimen to further mitigate cytokine release syndrome (CRS) incidence and severity and to evaluate SC elranatamab Q2W and Q4W dosing regimens in patients with relapsed/refractory MM. In part 1 of the study, patients will receive premedications and 2 step-up priming doses on cycle 1 D1 (4 mg) and D4 (20 mg), followed by elranatamab 76 mg QW for 6 cycles, and a dose Q2W thereafter if partial response or better is achieved and maintained. In part 2, higher doses of elranatamab ( > 76 mg) at increased intervals between doses will be evaluated. The primary endpoint is grade ≥2 CRS rate during cycle 1. Secondary endpoints include dose-limiting toxicities (part 2 only), objective response rate, duration of response, progression-free survival, overall survival, minimal residual disease negativity rate, safety, and pharmacokinetics. Eligible patients should be ≥18 years of age with a MM diagnosis and measurable disease according to IMWG criteria, refractory to at least one proteasome inhibitor, one immunomodulatory drug, and one anti-CD38 antibody, relapsed/refractory to their last treatment regimen, and have an ECOG performance status ≤1. Key exclusion criteria are smoldering MM, active plasma cell leukemia, amyloidosis, POEMS syndrome, stem cell transplant within 12 weeks of enrollment, active, uncontrolled bacterial, fungal, or viral infections, or any other active malignancy within 3 years prior to enrollment (except for adequately treated basal cell or squamous cell skin cancer, or carcinoma in situ), or previous treatment with an anti-BCMA bispecific antibody. The study is open at centers in the USA, UK, Japan, and Taiwan. Clinical trial information: NCT05014412.

A novel mutation at the AMEL primer binding region on the Y chromosome in AMELY negative male

Gender identification in forensic DNA typing is an important tool for criminal investigation as well as STR typing. Most methods are based on a size difference of amelogenin X and Y (AMELX and AMELY). There have been some reports that the method by amelogenin (AMEL) incorrectly typed some males as females because the AMELY allele is not detected. AMELY allele dropout is often caused by deletions encompassing AMELY on Yp11.2 and accompanied with Y-STR allele dropout (especially DYS458). However, an unusual deletion was found in our laboratory. The AMELY allele could be recovered by using another commercial kit and alternative AMEL primer sets, and the Y-STR markers resulted in a complete profile. Sequencing results showed that there was an 8bp deletion in AMELY at the position corresponding to 41-48bp downstream from the 3' end of the 6bp deletion site in AMELX. This is considered a novel mutation at a primer binding region.

Current laboratory protocols for detecting fish species with environmental DNA optimize sensitivity and reproducibility, especially for more abundant populations

AbstractAnalysing environmental DNA (eDNA) in seawater can aid in monitoring marine fish populations. However, the extent to which current methods optimize fish eDNA detection from water samples is unknown. Here, we test modifications to laboratory components of an eDNA metabarcoding protocol targeting marine finfish. As compared to baseline methods, amplifying a smaller proportion of extracted DNA yielded fewer species, and, conversely, amplifying a larger proportion identified more taxa. Higher-read species were amplified more reproducibly and with less variation in read number than were lower-read species. Among pooled samples, 20-fold deeper sequencing recovered one additional fish species out of a total of 63 species. No benefit was observed with additional PCR cycles, alternative primer concentrations, or fish-selective primers. Experiments using an exogenous DNA standard to assess absolute eDNA concentration suggested that, for a given proportion of a DNA sample, current laboratory methods for metabarcoding marine fish eDNA are near to maximally sensitive. Our results support the unofficial standard collection volume of one liter for eDNA assessment of commonly encountered marine fish species. We conclude that eDNA rarity poses the main challenge to current methods.

The invasive Korean bush mosquito Aedes koreicus (Diptera: Culicidae) in Germany as of 2020

BackgroundThe Korean bush mosquito Aedes koreicus was recently reported to have established a population in western Germany (Wiesbaden) in 2016. The species is difficult to distinguish morphologically from its close relative, the invasive Japanese bush mosquito Ae. japonicus, which is already widely distributed in many parts of Germany, including the area colonised by Ae. koreicus. Genetic confirmation of morphologically identified “Ae. japonicus” collection material, however, had only been done exceptionally before the German Ae. koreicus population became known.MethodsDried archived “Ae. japonicus” specimens both from the municipality of Wiesbaden and from deliberately and randomly selected distribution sites all over Germany were re-examined morphologically and genetically for admixture by Ae. koreicus. Moreover, cemeteries in the greater Wiesbaden area were sampled in 2019 and 2020 to check for Ae. koreicus spread. Korean and Japanese bush mosquitoes submitted to the German citizen science mosquito monitoring scheme “Mueckenatlas” in 2019 and 2020 were also subjected to particularly thorough species identification. The ND4 DNA sequences generated in this study in the context of species identification were phylogenetically compared to respective GenBank entries of Ae. koreicus. As a by-product, several genetic markers were evaluated for their suitability to identify Ae. koreicus.ResultsAedes koreicus specimens could be identified in mosquito collection material and submissions from Wiesbaden from 2015 onwards, suggesting establishment to have happened in the same year as Ae. japonicus establishment. Detections of Ae. koreicus from 2019 and 2020 in Wiesbaden indicate a negligible enlargement of the populated area as described for 2018. Two Ae. koreicus specimens were also submitted from the city of Munich, southern Germany, in 2019 but further specimens could not be identified during immediate local inspections. Comparison of ND4 sequences generated in this and other studies demonstrate a high degree of homology, suggesting that this DNA region is not informative enough for clarification of origins and relationships of Ae. koreicus populations. For genetic identification of Ae. koreicus, PCR primers used for classical CO1 barcoding were found to lead to mismatches and produce no or incorrect amplicons. Alternative CO1 primers or a validated ND4 marker should be used instead.ConclusionsAedes koreicus is probably introduced into Germany every now and then but rarely succeeds in becoming established. As with most European populations, the German population is characterised by a limited expansion tendency. Since Ae. koreicus is a potential vector, however, Asian bush mosquitoes found at new places should be examined quite carefully and known distribution areas of Ae. japonicus regularly checked for the presence of Ae. koreicus.Graphical