Hybridization Screening Research Articles

High prevalence of sexually transmitted infections and poor sensitivity and specificity of screening algorithms for Chlamydia and Gonorrhoea among female sex workers in Zimbabwe: Analysis of respondent-driven sampling surveys in 3 communities

Abstract Background: Effective strategies to reduce STI burden and transmission among female sex workers (FSW) and their networks are needed. We report STI prevalence among FSW in Zimbabwe and investigate the performance of screening algorithms. Methods: Respondent-driven sampling (RDS) surveys, including blood sampling for syphilis serology, were conducted among FSW in three communities in Zimbabwe in 2017. Additionally, a random sample of one third of participants were offered genital examination and STI testing. Data on symptoms and clinical signs were analysed to determine the proportion of asymptomatic and clinically inapparent STIs by HIV status, and the sensitivity, specificity and predictive values of syndromic, clinical and hybrid screening algorithms for chlamydia and gonorrhoea. Analyses were RDS-II weighted. Results: Overall, 2507 women were included in the RDS surveys and 661/836 (79.1%) of those randomly offered genital examination and STI testing accepted. STI prevalence by site ranged from 15.7%–20.0% for syphilis (RPR + TPHA), 6.8%–14.3% for gonorrhoea, 8.4%–10.1% for chlamydia, 26.6%–35.5% for trichomonas, and 37.0%–47.6% for any high-risk human papilloma virus. A high proportion of infections were both asymptomatic and clinically undetectable (gonorrhoea: 41.2%, chlamydia: 51.7%, trichomonas: 62.8%). Screening algorithms performed poorly whether based on symptoms only (sensitivity: 53.3% gonorrhoea, 43.3% chlamydia) or either symptoms or clinical signs (sensitivity: 58.7% gonorrhoea, 48.3% chlamydia). Conclusions: STI burden is high among FSW in Zimbabwe. The low sensitivity and specificity of screening algorithms used to guide syndromic management means that more effective approaches are required to strengthen STI control. As access to HIV-specific prevention methods like PrEP increase, support for consistent condom use need to be strengthened.

Design, Creation, and Biological Screening of Newer Coumarin-Coupled Heterocyclic Hybrids as Acetylcholinesterase Inhibitors that may be Useful in the Treatment of Alzheimer’s Disease

Design, Creation, and Biological Screening of Newer Coumarin-Coupled Heterocyclic Hybrids as Acetylcholinesterase Inhibitors that may be Useful in the Treatment of Alzheimer’s Disease

Autonomy and prevention: From conflicting to complementary aims of prenatal screening.

From an ethical point of view, there is an important distinction between two types of prenatal screening. The first of these targets maternal or foetal conditions (e.g., infectious diseases, blood group sensitization) where early detection allows for interventions that improve the chances of a healthy pregnancy outcome. The second screens for foetal conditions such as Down syndrome, where a timely diagnosis in most cases only allows for a choice between preparation for a child with special needs or termination of the pregnancy. Whereas the former makes an easy fit with the prevention aim of most other population screening programmes, the latter does not. In order to steer clear from a possible eugenic reading of its aim, a wide international consensus has emerged for the view that prenatal screening of this type should have the atypical aim of helping women (couples) to make autonomous reproductive choices, rather than reducing the birth prevalence of the relevant disorders. However, keeping these types of prenatal screening apart may become increasingly difficult given the development of tests, such as the Non-Invasive Prenatal Test, which cannot only be used for both types of screening but may also lead to interconnected findings on both sides of the divide. This makes it an urgent question: What the aim or aims of this new hybrid screening should be? As neither 'prevention' nor 'autonomy' will do, we argue for a normative framework that gives both aims their due, while recognizing the tensions between them.

Unraveling the interaction between a glycolytic regulator protein EhPpdk and an anaphase promoting complex protein EhApc10: yeast two hybrid screening, in vitro binding assays and molecular simulation study.

The anaphase promoting complex (APC or cyclosome) is a major ubiquitin ligase that coordinates mitotic and G1 progression, acting as a major regulator of chromosome segregation. While the human APC contains fourteen subunits, it is yet to be explored in the pathogen Entamoeba histolytica. Our study reveals the existence of a single functional Apc10 homolog in E. histolytica, which acts as a processivity factor of ubiquitin ligase activity in human. A cDNA library generated from HM1:IMSS strain of E. histolytica was screened for interaction partners of EhApc10 in yeast two hybrid study. The novel interactor, a glycolytic enzyme, pyruvate phosphate dikinase (Ppdk) was found to interact with EhApc10 and further validated by in vitro assay. A comprehensive in silico study has emphasized the structural and functional aspects, encompassing physicochemical traits, predictive 3D structure modelling, validation of EhApc10-EhPpdk interaction through molecular docking and simulation. The interplay between a cell cycle protein and a glycolytic enzyme highlights the connection between cellular metabolism and the cell cycle regulatory mechanism. The study serves as the groundwork for future research on the non-mitotic role of APC beyond cell cycle.

Design, Synthesis and Characterization of New Amide-Linked Chromone-Isoxazole Hybrids: In Vitro Anti-Bacterial and Antioxidant Evaluation, DFT Calculations, ADMET Profiling, Docking and Molecular dynamics simulation

In the present study, we report the synthesis, and biological evaluation of a new series of amide-linked chromone-isoxazole hybrids synthesized through a highly regioselective 1,3-dipolar cycloaddition (1,3-DC) reaction in a reproducible manner. The resulting products structures were identified by means of several spectroscopic techniques, including 13C NMR, 1H NMR, IR and MS spectrometry. In addition, the regio- and chemoselective synthesis of the newly synthesized chromone-isoxazole carboxamide hybrids were investigated via Density Functional Theory (DFT) calculations at the B3LYP/6-311G(d,p) level, which provided insights into molecular reactivity. Global and local reactivity indexes as well as the non-covalent interactions (NCI) analyses, and the findings explain the observed selectivity of the corresponding product. The antioxidant activity of all the new compounds was examined through the DPPH free radical scavenging assay, and the findings demonstrated a significant free radical scavenging power for the tested compounds. Notably, products (5c) and (5e) exhibited encouraging antioxidant effect. Antibacterial screening of the chromone-isoxazole hybrids against Escherichia coli, Porteus mirabilis, Bacillus subtilis and Staphylococcus aureus showed moderate activity, with compound 1 demonstrating the most promising antibacterial efficacy. To further explore the compound 1’s antibacterial potential, we employed molecular docking, dynamics simulations, ligand-protein contact analysis and ADMET profiling. Docking results revealed that compound 1 forms specific hydrogen bonds and hydrophobic interactions with bacterial targets 2W9S and 10FO, distinct from the interaction observed with standard antibacterial drug. Molecular dynamics simulation indicated stable protein-ligand conformations, despite slight ligand flexibility, and contact analysis identified a critical interaction stabilizing the complex. ADMET analysis confirmed that compound 1 possesses good drug-like properties, including oral bioavailability and low toxicity, indicating its potential as an effective antibacterial agent.

ГЕНЕТИЧЕСКИЙ СКРИНИНГ ПЕРСПЕКТИВНЫХ ГИБРИДОВ КАРТОФЕЛЯ РЕСПУБЛИКИ КОМИ

The studies were conducted with the aim of genetic screening of promising selection hybrids of potatoes to identify genotypes resistant to a complex of phytopathogens. The study was carried out at A.V.Zhuravskiy Institute of Agrobiotechnology of Komi Scientific Center of the Ural Branch of the Russian Academy of Sciences and Sintol company (Moscow) using the GenExpert reagent kit “Markers of potato resistance genes” (“Syntol”). For the study, 10 markers of genes encoding resistance to potato cancer, golden potato cyst nematode, pale potato cyst nematode, X-virus and Y-virus were selected (genes H1, Gro1-4 were detected using markers TG-689, 57R, N195 and Gro1-4-1, gene Sen1 – using marker NL25, gene Gpa2 – Gpa2-2). The objects of the study were leaves of plants of five potato hybrids undergoing selection testing in breeding nurseries: 2341-265 (Amur × Gala), 2339-8 (Oksania × Gala), 2339-9 (Oksania × Gala), 1992-14 (Udacha × Elmundo), 2000-60 (Colette × FZ 1867). The maximum number of markers (five in each) linked to resistance genes was revealed in samples 2339-8 and 1992-14: TG-689, 57R, N195 and NL25 in both hybrids; Gro1-4-1 marker in 2339-8 and Gpa2-2 in 1992-14. Only the marker of potato wart resistance gene, NL25, was noted in selection number 2341-265. Sample 2339-8 was characterized as a valuable source of R-genes, having in its genome the genes of resistance to golden potato cyst nematode (H1, Gro1-4) and potato wart (Sen1). The genotype of the 1992-14 hybrid, carrying the Gpa2 gene, is recommended for breeding new potato varieties resistant to various types of nematodes.

FATTY ACID DESATURASE4 enhances plant RNA virus replication and undergoes host vacuolar ATPase-mediated degradation

Abstract Emerging evidence indicates that fatty acid (FA) metabolic pathways regulate host immunity to vertebrate viruses. However, information on FA signaling in plant virus infection remains elusive. In this study, we demonstrate the importance of fatty acid desaturase (FAD), an enzyme that catalyzes the rate-limiting step in the conversion of saturated FAs into unsaturated FAs, during infection by a plant RNA virus. We previously found that the rare Kua-ubiquitin-conjugating enzyme (Kua-UEV1) fusion protein FAD4 from Nicotiana benthamiana (NbFAD4) was downregulated upon turnip mosaic virus (TuMV) infection. We now demonstrate that NbFAD4 is unstable and is degraded as TuMV infection progresses. NbFAD4 is required for TuMV replication, as it interacts with TuMV replication protein 6K2 and colocalizes with viral replication complexes. Moreover, NbFAD4 overexpression dampened the accumulation of immunity-related phytohormones and FA metabolites, and its catalytic activity appears to be crucial for TuMV infection. Finally, a yeast 2-hybrid library screen identified the vacuolar H+-ATPase component ATP6V0C as involved in NbFAD4 degradation and further suppression of TuMV infection. This study reveals the intricate role of FAD4 in plant virus infection, and sheds light on a new mechanism by which a V-ATPase is involved in plant antiviral defense.

WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

Formulation and optimization of copper selenide/PANI hybrid screen printing ink for enhancing the power factor of flexible thermoelectric generator: A synergetic approach

The stunning thermoelectric behavior of inorganic/organic hybrids and the lack of information on screen-printed flexible thermoelectric generators based on Cu3Se2 and Polyaniline for low-temperature applications have motivated this work. The synergic influence of various acid dopants, concentration, and annealing temperature of polyaniline on the power density of Cu3Se2/PANI ink-based flexible thermoelectric generator is explored. The presence of 1.5 wt% of H2SO4-PANI, annealed at 60 °C, decreased the bandgap and electrical resistance of Cu3Se2 by 11.96 % and 29.4 %, respectively. As a result, an increase of 46.06 % in the Seebeck coefficient and 242 % in the power output is achieved, which is many folds higher than the few reported works using novel materials. In addition, the Cu3Se2/H2SO4-PANI ink-based flexible thermoelectric generator with eight legs exhibited a maximum power output, power density, and power factor of 25.69 nW, 23.79 mW/m2, and 0.77 nW/m2K2 at ΔT = 100 °C, respectively under external load.

Hybrid Active and Passive Cable Contour Shielding of Magnetic Fields of Double-Circuit Overhead Power Lines

The purpose of the work is to design a hybrid screen designed to reduce the level of the magnetic field generated by double-circuit overhead power lines, to increase the efficiency of shielding the original magnetic field in residential buildings, to the level of sanitary standards and to reduce the sensitivity of the system to changes in system parameters. To achieve this goal, the structure of a hybrid screen was determined, consisting of a double contour active and a cable contour passive part. The developed hybrid screen is characterized by increased efficiency in reducing the magnetic field of industrial frequency. Designing a hybrid screen comes down to solving a minimax vector optimization problem, in which the vector objective function is calculated based on solution to Maxwell's equations in a quasi-stationary approximation using the COMSOL Multiphysics software package. The solution to the minimax vector optimization problem is calculated based on multi-particle swarm optimization algorithms from Pareto-optimal solutions taking into account binary preference relations. During the design of the hybrid screen, the coordinates of the spatial location of the two compensation windings, as well as the magnitude of the currents and its phases in the windings of the active screen, were calculated. The important results are theoretical and experimental studies on the effectiveness of the designed hybrid magnetic field shield generated by double-circuit overhead power lines. The significance of the results obtained lies in the fact that practical recommendations are given for a reasonable choice of the spatial arrangement of two shielding windings of a robust system of hybrid shielding of the magnetic field generated by double-circuit overhead power lines.

Preliminary screening of citrus hybrids for identifying tolerance to Phytophthora nicotianae

Phytophthora is one of the most devastating pathogens affecting citrus production. Breeding resistant genotypes is a durable strategy to manage this pathogen. In this study, in vitro screening of 31 citrus hybrids was done against Phytophthora nicotianae using detached leaf assay. The hybrids tested were developed in twoway crosses between Rangpur lime (RL, Phytophthora sensitive) and sour orange (SO, Phytophthora tolerant). The relative Phytophthora sensitivity was determined from the necrotic lesion area after three days of pathogen inoculation. The parents and hybrids could be differentiated into five resistance classes on the basis of necrotic lesion size and area. Among the 18 SO × RL (SR) hybrids, three hybrids viz. SR 3, SR 10 and SR 19 exhibited the least leaf necrotic length (2.8-3.7 mm), breadth (3.2-3.9 mm) and area (9.2-15.0 mm2), indicating a highly resistant reaction. From the 13 RL × SO (RS) hybrids, RS 6 and RS 22 showed highly resistant reaction by virtue of the least necrotic area (12.7-17.9 mm2). Twelve SR and four RS hybrids produced necrotic lesions of >216.8 mm2 area and were rated as highly susceptible in this assay. The parents- Rangpur lime and sour orange recorded highly susceptible and resistant reaction, respectively, which authenticated the results of the leaf assay. The five highly resistant hybrids identified in this study after ex vitro validation of Phytophthora tolerance should prove helpful in commercial citriculture.

Screening of Popular Maize Hybrids against fall Armyworm, Spodoptera frugiperda

Fall armyworm (Spodoptera frugiperda) was the member of noctuidae family which has got economic importance as polyphagous and extreme pest of many important crops, including maize in India and elsewhere. In this context, screening experiment was conducted by using 15 different maize cultivars viz., CP 818, NK 6240, GPMH 1110, GH 0727, 900 M Gold, NK 6801, SM- 2, S- 4717, GK 3207, KMH 225, KMH 8333, Sunny NMH 777, 30 B07, PMH 2244 and Kaveri minchu in MARS, Dharwad. The results revealed that PMH 2244 was recorded the least mean larval load (0.67 larvae per plant) compared to rest of the cultivars. Among fifteen different cultivars screened, Kaveri minchu recorded the highest mean leaf damage score (4.62) whereas, the hybrid PMH 224 recorded the least mean leaf damage score (0.73). The cultivars, PMH 2244, NK 6801 and 900 M Gold were recorded with the least egg mass (0.33 per plant) whereas, the hybrids SM- 2 and Kaveri minchu were recorded with the highest egg mass of 0.63 and 0.61 per plant, respectively. Biophysical parameters of plant such as plant height, leaf area, stem girth and trichome density have been implicated in maize resistance to the fall armyworm. All the plant characters except leaf area which are mentioned above showed significant negative correlation with fall armyworm infestation whereas, leaf area was found significantly positive correlation.

Experimental and computational evidence that Calpain-10 binds to the carboxy terminus of NaV1.2 and NaV1.6

Voltage-gated sodium channels (NaV) are pivotal proteins responsible for initiating and transmitting action potentials. Emerging evidence suggests that proteolytic cleavage of sodium channels by calpains is pivotal in diverse physiological scenarios, including ischemia, brain injury, and neuropathic pain associated with diabetes. Despite this significance, the precise mechanism by which calpains recognize sodium channels, especially given the multiple calpain isoforms expressed in neurons, remains elusive. In this work, we show the interaction of Calpain-10 with NaV's C-terminus through a yeast 2-hybrid assay screening of a mouse brain cDNA library and in vitro by GST-pulldown. Later, we also obtained a structural and dynamic hypothesis of this interaction by modeling, docking, and molecular dynamics simulation. These results indicate that Calpain-10 interacts differentially with the C-terminus of NaV1.2 and NaV1.6. Calpain-10 interacts with NaV1.2 through domains III and T in a stable manner. In contrast, its interaction with NaV1.6 involves domains II and III, which could promote proteolysis through the Cys-catalytic site and C2 motifs.

Additive manufacturing of sensor prototype based on 3D-extrusion-printed zirconia ceramics

Additive manufacturing of ceramics has attracted large interest due to its unique ability to rapidly prototype, low cost, and increased geometric complexity. Material extrusion technique is often considered for processing and shaping fine and dense ceramic structures because of the high solid loading in ink. In this work, 8 mol.% yttria-stabilized zirconia ink with 70 wt% ceramic loadings was prepared to print zirconia samples in horizontal and vertical directions, following two different filament orientations: 0/90° and ±45°. The study's main objective was to evaluate printing design influences on mechanical properties. This was assessed through flexural testing and digital image correlation analysis. Experimental findings showed that samples printed horizontally with ±45° filament orientation displayed the highest strength. A detailed inspection of fracture surfaces revealed that printing defects were the critical failure location sites formed after sintering and intimately related to printing design issues. After that, a conductive sensor was integrated into the surfaces of 3D-printed specimens by screen-printing silver-based conductive ink to analyze the structural health of zirconia samples. The sensing capabilities of printed conductive patterns were investigated using the four-point bending tests. The results demonstrated that the electrical resistance of the printed silver patterns increased as the applied load on zirconia substrates rose. It indicates that hybrid material extrusion and screen printing techniques are favored ways to manufacture sensors for structural detection of advanced 3D-printed ceramics.

Effectiveness of hybrid digital breast tomosynthesis/digital mammography compared to digital mammography in women presenting for routine screening at Maroondah BreastScreen: Study protocol for a co-designed, non-randomised prospective trial

BackgroundDigital breast tomosynthesis (DBT) for breast cancer screening has been shown in international trials to increase cancer detection compared with mammography; however, results have varied across screening settings, and currently there is limited and conflicting evidence on interval cancer rates (a surrogate for screening effectiveness). Australian pilot data also indicated substantially longer screen-reading time for DBT posing a barrier for adoption. There is a critical need for evidence on DBT to inform its role in Australia, including evaluation of potentially more feasible models of implementation, and quantification of screening outcomes by breast density which has global relevance. MethodsThis study is a prospective trial embedded in population-based Australian screening services (Maroondah BreastScreen, Eastern Health, Victoria) comparing hybrid screening comprising DBT (mediolateral oblique view) and digital mammography (cranio-caudal view) with standard mammography screening in a concurrent group attending another screening site. All eligible women aged ≥40 years attending the Maroondah service for routine screening will be enrolled (unless they do not provide verbal consent and opt-out of hybrid screening; are unable to provide consent; or where a ‘pushback’ image on hybrid DBT cannot be obtained). Each arm will enrol 20,000 women. The primary outcomes are cancer detection rate (per 1000 screens) and recall rate (percentage). Secondary outcomes include ‘opt-out’ rate; cohort characteristics; cancer characteristics; assessment outcomes; screen-reading time; and interval cancer rate at 24-month follow-up. Automated volumetric breast density will be measured to allow stratification of outcomes by mammographic density. Stratification by age and screening round will also be undertaken. An interim analysis will be undertaken after the first 5000 screens in the intervention group. DiscussionThis is the first Australian prospective trial comparing hybrid DBT/mammography with standard mammography screening that is powered to show differences in cancer detection. Findings will inform future implementation of DBT in screening programs world-wide and provide evidence on whether DBT should be adopted in the broader BreastScreen program in Australia or in subgroups of screening participants.

Characterizing research partnerships in child health research: A scoping review.

Research partnerships between researchers and knowledge users (KUs) in child health are understudied. This study examined the scope of KU engagement reported in published child health research, inclusive of health research partnership approaches and KU groups. Search strategies were developed by a health research librarian. Studies had to be in English, published since 2007, and were not excluded based on design. A two-step, multiple-person hybrid screening approach was used for study inclusion. Data on study and engagement characteristics, barriers and facilitators, and effects were extracted by one reviewer, with 10% verified by a second reviewer. Three hundred fifteen articles were included, with 243 (77.1%) published between 2019 and 2021. Community-based participatory research was the most common approach used (n = 122, 38.3%). Most studies (n = 235, 74.6%) engaged multiple KU groups (range 1-11), with children/youth, healthcare professionals, and parents/families being most frequently engaged. Reporting of barriers and facilitators and effects were variable, reported in 170 (53.8%) and 197 (62.5%) studies, respectively. Publications have increased exponentially over time. There is ongoing need to optimize evaluation and reporting consistency to facilitate growth in the field. Additional studies are needed to further our understanding of research partnerships in child health.

Screening of castor hybrids and their parents against wilt disease

Castor is an industrially important non-edible oilseed crop widely cultivated in the arid and semi-arid regions of the world. Wilt (Fusarium oxysporum f. sp. ricini) disease has become a serious problem in India, though wilt resistant hybrids viz., GCH-4, GCH-5, GCH-7, GCH-8, GCH-9 and GCH 10 have been released. So investigation on screening of castor hybrids and their parents against wilt disease was undertaken under poly house conditions. The results revealed that out of six, three parent's viz., SKP-84, JI-368 and 48-1 were resistant, while two hybrids i.e. SKP-84 x JI- 368 and GEETA x SH-72 were found resistant to wilt disease. Parent GEETA found moderately resistant, whereas susceptibility for the disease was observed in parents viz., VP-1, VI-9, SH-72 and JI-35 and Hybrid VP-1 x VI-9 reported susceptibility to wilt disease. Our findings confirmed the importance of selecting the parents for the development of resistant hybrids in disease-resistance breeding programmes.

Analysis of deafness susceptibility gene of neonates in northern Guangdong, China

This study aimed to explore the molecular epidemiology characteristics of deafness susceptibility genes in neonates in northern Guangdong and provide a scientific basis for deafness prevention and control. A total of 10,183 neonates were recruited between January 2018 and December 2022 at Yuebei People's Hospital. Among these, a PCR hybridization screening group of 8276 neonates was tested for four deafness genes: GJB2, SLC26A4, mtDNA, and GJB3 by PCR hybridization. Another group used next-generation sequencing (NGS) to detect genetic susceptibility genes in 1907 neonates. In PCR hybridization screening group, 346 (4.18%) of 8276 neonates were found to be carriers of the deafness gene. Among these, 182 (2.2%) had GJB2 variants, 114 (1.38%) had SLC26A4 variants, 35 (0.42%) had mtDNA variants, and 15 (0.18%) had GJB3 variants. In NGS Screening Group, 195 out of 1907 neonates were found to be carriers of the deafness gene, with a positive rate of 10.22%. Among these, 137 (7.18%) had GJB2 variants, 41 (2.15%) had SLC26A4 variants, 11 (0.58%) had mtDNA variants, and 6 (0.31%) had GJB3 variants. The prevalence of deafness gene variants was high in Northern Guangdong Province. The most common gene for deafness was GJB2, followed by SLC26A4 and mtDNA. GJB3 variants are rare. Compared with PCR hybridization method, NGS technology can expand the screening scope and greatly improve the detection rate of deafness genes. The c.109G>A of GJB2 was found to occur at a high frequency, which should be considered. Therefore, it is important to conduct neonatal deafness gene screening to prevent and control hereditary deafness.

Screening of different cotton hybrids containing Bt genes against Pectinophora gossypiella (Saunders)

Screening of different cotton hybrids containing Bt genes against Pectinophora gossypiella (Saunders)

Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.