BACKGROUND: The digital era has witnessed an escalating dependence on digital platforms for news and information, coupled with the advent of \"deepfake\" technology. Deepfakes, leveraging deep learning models on extensive data sets of voice recordings and images, pose substantial threats to media authenticity, potentially leading to unethical misuse such as impersonation and the dissemination of false information.
OBJECTIVE: To counteract this challenge, this study aims to introduce the concept of innate biological processes to discern between authentic human voices and cloned voices. We propose that the presence or absence of certain perceptual features, such as pauses in speech, can effectively distinguish between cloned and authentic audio.
METHODS: A total of 49 adult participants representing diverse ethnic backgrounds and accents were recruited. Each participant contributed voice samples for the training of up to 3 distinct voice cloning text-to-speech models and 3 control paragraphs. Subsequently, the cloning models generated synthetic versions of the control paragraphs, resulting in a data set consisting of up to 9 cloned audio samples and 3 control samples per participant. We analyzed the speech pauses caused by biological actions such as respiration, swallowing, and cognitive processes. Five audio features corresponding to speech pause profiles were calculated. Differences between authentic and cloned audio for these features were assessed, and 5 classical machine learning algorithms were implemented using these features to create a prediction model. The generalization capability of the optimal model was evaluated through testing on unseen data, incorporating a model-naive generator, a model-naive paragraph, and model-naive participants.
RESULTS: Cloned audio exhibited significantly increased time between pauses (P<.001), decreased variation in speech segment length (P=.003), increased overall proportion of time speaking (P=.04), and decreased rates of micro- and macropauses in speech (both P=.01). Five machine learning models were implemented using these features, with the AdaBoost model demonstrating the highest performance, achieving a 5-fold cross-validation balanced accuracy of 0.81 (SD 0.05). Other models included support vector machine (balanced accuracy 0.79, SD 0.03), random forest (balanced accuracy 0.78, SD 0.04), logistic regression, and decision tree (balanced accuracies 0.76, SD 0.10 and 0.72, SD 0.06). When evaluating the optimal AdaBoost model, it achieved an overall test accuracy of 0.79 when predicting unseen data.
CONCLUSIONS: The incorporation of perceptual, biological features into machine learning models demonstrates promising results in distinguishing between authentic human voices and cloned audio.

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral replication in infected cell. One such gene is deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also known as UL50. In this study, we analyzed the amino acid sequence of UL50 (dUTPase) using bioinformatics tools and found that it was highly conserved among herpesvirus family. Then, it was cloned and expressed in Escherichia coli Rosetta (DE3), induced by isopropy1-b-D-thiogalactopyranoside (IPTG) and the recombinant UL50 protein was purified to immunize rabbits for the preparation of polyclonal antiserum. The results indicated that the UL50 gene of BoHV-1 was composed of 978 nucleotides, which encoded 323 amino acids. Western blot analysis revealed that polyclonal sera against UL50 reacted with a band of 34 kDa. Furthermore, immunofluorescence assay showed that UL50 localized in the cytoplasmic area. Taken together, UL50 was successfully cloned, expressed and detected in BoHV-1-infected cells and was localized in the cytoplasm to help in the replication of BoHV-1 in infected cells.

Xist is an X-linked ribonucleic acid (RNA) gene responsible for the cis induction of X chromosome inactivation (XCI). In cloned mammalian embryos, Xist is ectopically activated at the morula to blastocyst stage on the X chromosome that is supposed to be active, thus resulting in abnormal XCI. Suppression of erroneous Xist expression by injecting small interfering RNA (siRNA) remarkably increased the developmental efficiency of cloned male mouse embryos by approximately 10-fold. However, injection of anti-Xist siRNA resulted in only a slight increase in the developmental ability of injected cloned male pig embryos because the blocking effect of the injected siRNA was not maintained beyond the morula stage, which is 5 days post-activation. To develop a more effective approach for suppressing the ectopic expression of Xist in cloned pig embryos, we compared the silencing effect of short hairpin RNA (shRNA) and siRNA on Xist expression and the effects of these two Xist knockdown methods on the developmental competence of cloned male pig embryos. Results indicated that an shRNA-based RNA interference (RNAi) has a longer blocking effect on Xist expression than an siRNA-mediated RNAi. Injection of anti-Xist shRNA plasmid into two-cell-stage cloned male pig embryos effectively suppressed Xist expression, rescued XCI at the blastocyst stage, and improved the in vitro developmental ability of injected cloned embryos. These positive effects, however, were not observed in cloned male pig embryos injected with anti-Xist siRNA. This study demonstrates that vector-based rather than siRNA-mediated RNAi of Xist expression can be employed to improve pig cloning efficiency.

Cloned pig fetuses produced by somatic cell nuclear transfer show a high incidence of erroneous development in the uteri of surrogate mothers. The mechanisms underlying the abnormal intrauterine development of cloned pig fetuses are poorly understood. This study aimed to explore the potential causes of the aberrant development of cloned pig fetuses. The levels of numerous fatty acids in allantoic fluid and muscle tissue were lower in cloned pig fetuses than in artificial insemination-generated pig fetuses, thereby suggesting that cloned pig fetuses underwent fatty acid deficiency. Cloned pig fetuses also displayed trophoblast hypoplasia and a reduced expression of placental fatty acid transport protein 4 (FATP4), which is the predominant FATP family member expressed in porcine placentas. This result suggested that the placental fatty acid transport functions were impaired in cloned pig fetuses, possibly causing fatty acid deficiency in cloned pig fetuses. The present study provides useful information in elucidating the mechanisms underlying the abnormal development of cloned pig fetuses.

The reproductive cloning in buffalo in India has been started using a simplified somatic cell nuclear transfer technique named handmade cloning. Since the birth of first cloned female buffalo in 2009, a number of buffalo clones have been produced in India by utilizing different types of donor cells such as ear cells, embryonic stem cells, semen somatic cells and urine somatic cells. The use of buffalo cloning on a large scale is restricted due to low pregnancy rates and poor calf survival. Considerable attempts have been made to improve the overall buffalo cloning efficiency, particularly by modifying epigenetic reprogramming of cloned embryos. Previous studies have demonstrated that chemical epigenetic modifiers such as trichostatin A and 5-aza-2\'-deoxycytidine, m-carboxycinnamic acid bishydroxamide can be used to treat donor somatic cells and reconstructed fused embryos to correct the epigenetic reprogramming to enhance the overall cloning efficiency in terms of live birth rates.

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.

The extremely low full-term developmental efficiency of cloned pig embryos limits the practical application of pig cloning techniques. Maternal dietary supplementation of the nutritionally important amino acid, arginine, can enhance prenatal developmental rate of in vivo fertilization-derived pig embryos. It was hypothesized that maternal dietary addition of arginine can also improve the developmental capacity of cloned pig embryos. To test this hypothesis, there was a comparison of the reproductive performance between recipient sows fed an L-arginine-supplemented diet (L-Arg group) and those fed the control diet (control group). There was a subsequent comparison of the developmental indexes of cloned piglets farrowed in the L-Arg and control groups of surrogate sows. Dietary supplementation of L-arginine during gestation days 14-75 increased the plasma concentrations of arginine and arginine metabolites, including nitric oxide, spermidine, and putrescine in recipient sows of transferred cloned pig embryos. Although maternal arginine addition did not affect the birth weight and placental development indexes of newborn cloned piglets, it significantly increased the ratio of total cloned piglets born to total transferred cloned pig embryos by increasing the pregnancy rate of recipient sows. The results of this study suggest that nutritional management of recipient sows is an effective approach to improve the developmental rate of cloned pig embryos.

Wuzhishan miniature pig is one of the four most important pig breeds in China and has many major economic characteristics. Herein, we successfully used SCNT to clone Wuzhishan miniature pig. First, ear fibroblasts were isolated from a 2-year-old female Wuzhishan miniature piglet to be used as the donor cell. Second, good-quality COCs were selected from ovaries obtained from pigs at a local slaughterhouse and cultured. Mature eggs with the first polar body and ear fibroblasts were applied SCNT. Lastly, we in total produced 12 piglets with 7 piglets surviving to adults. Next, we used these pigs to test alloxan toxicity and to build T I D diabetes type. We know that diabetes mellitus is a chronic heterogeneous metabolic disease characterized by a high blood glucose level and abnormal insulin secretion. In this study, T I D (type I diabetes) was experimentally induced in cloned Wuzhishan miniature pigs with alloxan. In brief, an intravenous injection of alloxan (group B: 170 mg/kg, n = 3) was administered to pigs weighing between 27 and 39 kg. Sterile saline was administered to control pigs (n = 3). We determined the glycometabolism related index, performed an intravenous glucose tolerance test, and carried out immunohistochemistry experiments. There were no significant differences in body weight, blood glucose, and serum insulin in all groups, before treatment. The level of blood glucose was significantly higher (P < 0.05) in group B (12.18 ± 0.70 mmol/L) than in the control (2.93 ± 0.39 mmol/L). By contrast, the level of serum insulin was lower in group B (5.641 ± 0.573 μIU/mL) than in the control (7.578 ± 0.539 μIU/mL). Histological studies by hematoxylin and eosin (H&E) revealed a loss of β-cells in the pancreas from pigs treated with 170 mg/kg alloxan. Immunolocalization studies showed a decrease in insulin reactivity in this treatment group as well. To conclude, our model holds promise in future studies of diabetes drug testing and islet xenotransplantation.