ORF8 protein of SARS-CoV-2 reduces male fertility in mice.

As one of the most rapidly evolving proteins of the genus Betacoronavirus, open reading frames (ORF8's) function and potential pathological consequence in vivo are still obscure. In this study, we show that the secretion of ORF8 is dependent on its N-terminal signal peptide sequence and can be inhibited by reactive oxygen species scavenger and endoplasmic reticulum-Golgi transportation inhibitor in cultured cells. To trace the effect of its possible in vivo secretion, we examined the plasma samples of coronavirus disease 2019 (COVID-19) convalescent patients and found that the patients aged from 40 to 60 had higher antibody titers than those under 40. To explore ORF8's in vivo function, we administered the mice with ORF8 via tail-vein injection to simulate the circulating ORF8 in the patient. Although no apparent difference in body weight, food intake, and vitality was detected between vehicle- and ORF8-treated mice, the latter displayed morphological abnormalities of testes and epididymides, as indicated by the loss of the central ductal lumen accompanied by a decreased fertility in 5-week-old male mice. Furthermore, the analysis of gene expression in the testes between vehicle- and ORF8-treated mice identified a decreased expression of Col1a1, the loss of which is known to be associated with mice's infertility. Although whether our observation in mice could be translated to humans remains unclear, our study provides a potential mouse model that can be used to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the human reproductive system.

Changes in anisometropia by age in children with hyperopia, myopia, and antimetropia.

Anisometropia is a unique condition of both eyes and it is associated with vision problems such as amblyopia and reduced stereoacuity. Previous studies have not reported its change pattern by age and its correlation with the refractive condition of both eyes. This study aims to compare the changes in anisometropia by age in children with hyperopia, myopia, and antimetropia. In total, 156 children were included. Children aged 3-11 years with anisometropia ≥ 1.00 D were followed up for ≥ 1 year with ≥ 2 visits at two medical centers in Taiwan. Refractive errors by cycloplegic autorefractometry, best-corrected visual acuity, eye position, and atropine use were recorded. The children were divided into hyperopic, myopic, and antimetropic groups. The results showed that anisometropia decreased in children aged < 6 years (3.34-2.96 D; P = 0.038) and increased in older children (2.16-2.55 D; P = 0.005). In children aged 3, 4, 5, and 6 years, the mean anisometropia was higher in children with myopia and antimetropia than in those with hyperopia (P = 0.005, 0.002, 0.001, and 0.011, respectively). The differences were not significant in children aged > 6 years (all P > 0.05). The factors associated with changes in anisometropia were age, refractive group, amblyopia, and strabismus. Anisometropia decreased with age in children younger than 6 years, and the changes in anisometropia was found in children with myopia and antimetropia.

Auricular acupressure for adverse events following immunization after COVID-19 vaccine injection: A multicentre, blinded, randomized controlled trial.

BACKGROUND: Some adverse events following immunization (AEFI) were observed in potential corelation with COVID-19 vaccination but without prevention or ongoing trial for it. We aimed to investigate efficacy of auricular acupressure (AuriAc) therapy in preventing AEFI after first dosage of the vaccine. METHODS: We performed a multicentre randomized controlled trial with three arms, including AuriAc, SAuriAc (sham auricular acupressure), and TrAsU (treatment as usual) group, carried out in four medical institutions in Chengdu, China, from March 17th to April 23rd, 2021. We enrolled participants based on eligibility criteria and randomized them into three groups: AuriAc (AEFI-specific auricular points applied, n = 52), SAuriAc (n = 51) or TrAsU (n = 44) group. Primary outcomes were percentages of any AEFI and local pain, and secondary outcomes were percentages who reported other AEFI. They were followed at 1, 3, 5, 7, and 14 days, by phone or online, with severity evaluated. RESULTS: 147 participants (73.47% females) were included with median age as 31 years (25-45, IQR). One day after the injection, participants in AuriAc group reported significant reduction on percentages of any AEFI [intention-to-treat, difference of percentage (DP) = -20.13, 95%CI: - 0.39, - 0.02, p = 0.01; per-protocol, DP = -22.21, 95%CI: - 0.40, - 0.03, P = 0.02] and local pain (per-protocol, DP = -18.40, 95%CI: -0.36, -0.01, P = 0.04), compared with TrAsU group. The effects were slight at other follow-up days and for other outcomes, and with a low percentage of mild local allergic reactions. CONCLUSIONS: We firstly explored potential of AuriAc for preventing AEFI related to COVID-19 vaccine injection, which is beneficial for the vaccine recipients, but evidence is limited. TRIAL REGISTRATION: chictr.org.cn no. ChiCTR2100043210 (http://www.chictr.org.cn/showproj.aspx?proj=121519).

Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication.

BACKGROUND: Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. RESULTS: Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. CONCLUSIONS: Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery.

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.

Auricular acupressure for adverse events following immunization related to COVID-19 vaccine injection: study protocol for a multicenter, three-arm, blinded randomized controlled trial.

BACKGROUND: Some pain, fatigue, and gastrointestinal adverse events were observed in potential association with injection of COVID-19 vaccines, while there was no preventive intervention for it. We aim to investigate the efficacy of auricular acupressure (AA) therapy in preventing and relieving AEFI after injection of COVID-19 vaccine. METHODS: The study design is a randomized, multicentre, three-arm controlled, single-blind trial. Participants meeting the inclusion criteria will be advertised and enrolled and assigned in the medical institutions randomly for post-injection observation. No less than 360 participants will be randomized into one of three groups: auricular acupressure group, sham auricular acupressure group, and wait-list group. Interventions will be performed immediately and will happen 4 to 5 times per day for 5 days. The primary clinical outcomes will be quality and quantity evaluation among participants who reported any AEFI and who reported local pain at injection site. Secondary outcomes will concern headache, muscle and (or) joint pain, fatigue, nausea, vomiting, diarrhoea, and other potential events. All the outcomes will be assessed at baseline and 1, 3, 5, 7, and 14 days after the injection. Both intention-to-treat and per-protocol analyses will be performed, with significance level determined as 5%. DISCUSSION: Results of this trial will help to clarify the value of auricular acupressure therapy in preventing and relieving overall and certain adverse events following immunization after injection of COVID-19 vaccine. TRIAL REGISTRATION: China Clinical Trial Registry (ChiCTR) ( ChiCTR2100043210 ). Registered on 8 February, 2021.

Traditional Chinese medicine auricular point acupressure for the relief of pain, fatigue, and gastrointestinal adverse reactions after the injection of novel coronavirus-19 vaccines: a structured summary of a study protocol for a multicentre, three-arm, single-blind, prospective randomized controlled trial.

OBJECTIVES: To investigate if traditional Chinese medicine (TCM) auricular point acupressure (APA) can alleviate and (or) reduce the pain (including injection site pain, headache, other muscle and joint pain), fatigue, and gastrointestinal adverse reactions (including nausea, vomiting, diarrhea), after the injection of novel coronavirus-19 vaccines (NCVs). TRIAL DESIGN: The study is designed as a multicentre, parallel-group, three-arm, single-blind, prospective, randomized (1:1:1 ratio) study. PARTICIPANTS: More than 360 participants will be recruited from healthy people who vaccinate NCVs in 5 community healthcare centres in the Sichuan province of China and 1 university hospital (Hospital of Chengdu University of Traditional Chinese Medicine). INCLUSION CRITERIA: ①Vaccinators meets the conditions of NCVs injection and have no contraindications to it. The details shall be subject to the instructions of the NCVs used and the statement of medical institutions. The first dose of NCVs injection shall be completed within 24 hours from the time of injection to the time of enrolment; ②No redness, swelling, injury or infection of the skin or soft tissue of both ears, which is not suitable for APA; ③No history of alcohol and adhesive tape contact allergy; ④18-59 years old, regardless of gender; ⑤Those who were able to complete the questionnaire independently at the time of the first and second dose of NCVs and on the 3rd, 7th and 15th day after the first and second dose of NCVs respectively; ⑥Those who agree to participate in the trial and sign the informed consent, and can seriously abide by the precautions after the injection of NCVs and the requirements of traditional Chinese medicine auricular point plasters sticking and acupressure. EXCLUSION CRITERIA: ①Those who are not suitable to be vaccinated because they belong to the contraindication or cautious population; ②Those who have participated in other clinical trials within 4 weeks before the start of this study; ③No chronic/habitual/persistent headache, Muscle or joint pain, fatigue, diarrhea, nausea, retching or vomiting before the injection of NCVs, and no related diseases present (details of this item is listed in full protocol); ④Those who are in use or have received TCMAPA within 2 weeks before the trial; ⑤Pregnant or lactating women; ⑥Participants with other serious primary diseases and psychosis. INTERVENTION AND COMPARATOR: ①Auricular point acupressure group: participants receive bilateral, symptom-specific TCMAPA in 5 auricular points (per side, 10 points bilateral) for 5 days, 3-4 times (about 1 min each time) of self-acupressure per day, after each NCVs injection (10 days in total). ②Sham auricular point acupressure group: participants receive bilateral, none symptom-specific, sham APA in 5 auricular points (per side, 10 points bilateral) for 5 days, 3-4 times (about 1 min each time) of self-acupressure per day, after each NCVs injection (10 days in total). ③Blank control group: Non-intervention blank control. The Hebei medical device Co. Ltd, Hebei, China manufactured the auricular point sticking plasters. MAIN OUTCOMES: Primary outcomes are all scores of visual analogue scale (VAS) based on subjective judgment of the participants included, including VAS score of pain at injection site, headache, muscle and joint pain, fatigue, nausea, retching, vomiting and diarrhea. Time points for outcomes above are the same: ①Immediately after first and second injection of the vaccine (Baseline assessment); ②Three days after first and second injection of the vaccine; ③Seven days after first and second injection of the vaccine; ④Fifteen days after first and second injection of the vaccine. RANDOMISATION: Participants will be randomized in 1:1:1 ratio to each group by computerized random number generator, and independently in each sub-centre. BLINDING (MASKING): Participants, information collectors and statistical evaluators will be blinded between APA group and sham APA group. No blinding in the control group. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): No less than 360 participants will be randomized in 1:1:1 ratio to each group. TRIAL STATUS: Protocol version 2.0 of February 3rd, 2021. Recruitment is expected to start on February 18th, 2021, and to finish on March 12th, 2021. TRIAL REGISTRATION: This trial was registered in the China Clinical Trial Registry (ChiCTR) ( ChiCTR2100043210 ) on 8th February, 2021. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Fabrication and characteristics of highly [Formula: see text]-oriented nanotwinned Au films.

Fine grained and nanotwinned Au has many excellent properties and is widely used in electronic devices. We have fabricated [Formula: see text] preferred-oriented Au thin films by DC plating at 5 mA/cm2. Microstructure analysis of the films show a unique fine grain structure with a twin formation. Hardness tests performed on electroplated [Formula: see text] Au films show a hardness 47% greater than random and untwinned Au. We then achieved direct bonding between two Au [Formula: see text] surfaces operating at 200 °C for an hour in a vacuum oven. The highly-oriented [Formula: see text] nanotwinned Au films could be an ideal material in many gold products.

Low-temperature direct copper-to-copper bonding enabled by creep on (111) surfaces of nanotwinned Cu.

Direct Cu-to-Cu bonding was achieved at temperatures of 150-250 °C using a compressive stress of 100 psi (0.69 MPa) held for 10-60 min at 10(-3) torr. The key controlling parameter for direct bonding is rapid surface diffusion on (111) surface of Cu. Instead of using (111) oriented single crystal of Cu, oriented (111) texture of extremely high degree, exceeding 90%, was fabricated using the oriented nano-twin Cu. The bonded interface between two (111) surfaces forms a twist-type grain boundary. If the grain boundary has a low angle, it has a hexagonal network of screw dislocations. Such network image was obtained by plan-view transmission electron microscopy. A simple kinetic model of surface creep is presented; and the calculated and measured time of bonding is in reasonable agreement.

Effect of grain orientations of Cu seed layers on the growth of <111>-oriented nanotwinned Cu.

We investigate the growth of Cu films on two different Cu seed layers: one with regular <111>-oriented grains and the other with very strong <111>-preferred orientation. It is found that densely-packed nanotwinned Cu (nt-Cu) can be grown by pulsed electroplating on the strong <111>-oriented Cu seed layer without a randomly-oriented transition layer between the nt-Cu and the Cu seed layer. The electroplated nt-Cu grow almost epitaxially on the seed layer and formed <111>-oriented columnar structures. However, with the regular <111>-oriented Cu seed, there is a randomly-oriented transition layer between the nt-Cu and the regular <111>-oriented Cu seed. The results indicate that the seed layer plays a crucial role on the regularity of <111>-oriented nanotwinned Cu.

Unidirectional growth of microbumps on (111)-oriented and nanotwinned copper.

Highly oriented [111] Cu grains with densely packed nanotwins have been fabricated by direct-current electroplating with a high stirring rate. The [111]-oriented and nanotwinned Cu (nt-Cu) allow for the unidirectional growth of Cu(6)Sn(5) intermetallics in the microbumps of three-dimensional integrated-circuit packaging; a uniform microstructure in a large number of microbumps of controlled orientation can be obtained. The high-density twin boundaries in the nt-Cu serve as vacancy sinks during the solid-state reaction between Pb-free solder and Cu and greatly reduce the formation of Kirkendall (or Frenkel) voids.