Passage Level Research Articles

Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.

Isolation and genetic characterization of parvoviruses from domestic cats reveals emergence of CPV-2c in India: A first report.

The objective of the present study was to isolate and characterize the VP2 gene of parvoviruses from domestic cats in India. For that, 38 fecal samples were screened by PCR with 36.84% positivity. Sequence analysis of those isolates showed canine parvovirus type-2c (CPV-2c) as the predominant variant, followed by feline panleukopenia virus (FPV) and 2a. Phylogenetic analysis of the CPV-2c sequences revealed clustering with Singaporean, South Korean, Mongolian and Bangladeshi dog 2c sequences. Phylogenetic analysis of the 2a isolate (MZC 2) was found to be clustered with Indian, Thai and Singaporean dog 2a isolates. Similarly, all the four FPV sequences were ancestrally related to Indian dog and cat FPV sequences hinting towards interspecies transmission between dogs and cats. Both synonymous and non-synonymous mutations were evident in CPV-2c, 2a and FPV sequences indicative of active evolution. In cell culture medium, CPV-2 showed cytopathogenic effects at the third passage level. In conclusion, the study provided the first report of CPV-2c in cats from India, which demands for extensive epidemiological surveillance to monitor interspecies spread and to shed more light on viral phylogenomics, their distribution in the country and in the Southeast Asian region and usage of current vaccines.

The Cytokine Levels of Cord Blood- and Wharton's Jelly-Derived Mesenchymal Stem Cells from Early to Late Passages.

Mesenchymal stem cells (MSCs) are promising for clinical studies owing to their self-renewal, multipotency, trophic, and immunomodulatory properties. This study aimed to investigate the cytokine levels of human umbilical cord blood (CB) and Wharton's Jelly-(WJ) derived MSCs relevant to immune modulation on different passage levels in vitro. Umbilical CB MSCs were isolated using the ficoll-paque gradient method, and WJ-MSCs were isolated by the explant method. After isolation, the MSCs were characterized using flow cytometry. The supernatant cytokine levels (interferon-gamma (IFN-γ), interleukin 4 (IL-4), interleukin 17 (IL-17)) of MSCs at each passage were evaluated using the ELISA assay. MSCs exhibited different cytokine levels with each passage number. In WJ-MSC culture supernatants, IL-17 levels significantly increased at P4 and P5 compared to the first passage (p < 0.005), while the other passages showed a decrease. IFN-γ levels increased at passage P1 and P4 and decreased at other passages (p < 0.005). IL-4 levels significantly increased only at passage P3 (p < 0.005). In CB-MSC culture supernatants, IL-17 and IL-4 cytokines decreased compared to P0, while IFN-γ cytokine increased from P0 (p < 0.005). The changing ratio of cytokine levelsfor both CB-MSCs and WJ-MSCs were similarly maintained from early to late passages. More research is needed to understand the immunomodulatory functions of MSCs.

التكرار الصوتي في شعر بدوي الجبل

The research seeks to study one of the important phenomena of the Arabic language, which is the phenomenon of vocal repetition in the poetry of Badawi al-Jabal. It is no secret that the voice has great importance in Arabic, as language is nothing but “sounds with which every people expresses their purposes,” and repetition is a distinctive stylistic phenomenon in poetry. Semantic value and condensing energies that complete the images and perform their suggestive function in a consistent manner with the general purpose intended by the speaker. It is also considered one of the most prominent stylistic phenomena for understanding the literary text. Its aesthetics are highlighted in the artistic text through alternating words and repeating them in the context of poetic expression, and forming a melody. Musically, it is intended by the composer in his poetry or prose, as repetition highlights something important to the poet that he reveals by repeating it, in addition to the fact that repetition is one of the tools that increase the beauty and beauty of the text. This study has clarified the meaning of phonetic repetition in language and terminology and the opinions of ancient and modern Arabic scholars regarding it, and its most important benefits in the poetic text - in the collection of the poet Badawi Al-Jabal - such as emphasis, increased alertness, and repetition of the related to negate heedlessness and others, in addition to mentioning the most important levels of repetition, which are repetition at the level of the letter and at the letter level. Word level, sentence level, and passage level. The researcher relied on a descriptive and analytical approach, and the study concluded with a conclusion that included the most important results reached by the researcher.

Utilizing passage‐level relevance and kernel pooling for enhancing BERT‐based document reranking

AbstractThe pre‐trained language model (PLM) based on the Transformer encoder, namely BERT, has achieved state‐of‐the‐art results in the field of Information Retrieval. Existing BERT‐based ranking models divide documents into passages and aggregate passage‐level relevance to rank the document list. However, these common score aggregation strategies cannot capture important semantic information such as document structure and have not been extensively studied. In this article, we propose a novel kernel‐based score pooling system to capture document‐level relevance by aggregating passage‐level relevance. In particular, we propose and study several representative kernel pooling functions and several different document ranking strategies based on passage‐level relevance. Our proposed framework KnBERT naturally incorporates kernel functions from the passage level into the BERT‐based re‐ranking method, which provides a promising avenue for building universal retrieval‐then‐rerank information retrieval systems. Experiments conducted on two widely used TREC Robust04 and GOV2 test datasets show that the KnBERT has made significant improvements over other BERT‐based ranking approaches in terms of MAP, P@20, and NDCG@20 indicators with no extra or even less computations.

Application of Novel Gill Cell Line from Lates calcarifer for Recognizing Metals Using Probes.

Lates calcarifer (Bloch) is a potential candidate fish species for culture in marine and brackishwater. A continuous gill cell line was derived from L. calcarifer by the explant culture method and has been passaged for 132 times, in Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS) at 28°C. The cells showed a rate of recovery between 90 and 95% after being successfully cryopreserved at various passage levels and formed monolayer in 2-3days without any morphological changes. Immunophenotypic analysis of the SBG cell line revealed that they are of epithelial origin. Polymerase chain reaction assay using mitochondrial 12S rRNA primer specific to L. calcarifer was used to confirm the authenticity of the established gill cell line origin from seabass. The transfection efficiency was evaluated in Seabass Gill (SBG) cell line using pEGFP-N1 and Lipofectamine™ 3000. Transfection efficiency was found to be between 13 and 16%. The cytotoxicity of three different metal detecting probes was evaluated by MTT and Alamar blue assays to determine safe concentration. The result revealed that SBG cell line can be applied for recognition of metals using probes. The current study established, for the first time, a gill-derived cell line (SBG) from Lates calcarifer and its application for the detection of intracellular indium, mercury, and lutetium ions by specific fluorescent probes.

The Relationships of Oral Reading Fluency at Word, Sentence, and Passage Levels and Reading Comprehension in Chinese.

This study aims to expand our understanding of the relations of oral reading fluency at word, sentence, and passage levels to reading comprehension in Chinese-speaking secondary school-aged students. In total, 80 participants (46 males and 34 females) ranging from 13 to 15 years old joined this study and were tested on tasks of oral reading fluency at three levels, reading comprehension, and nonverbal IQ. Our results showed a clear relationship from fluency at the level of the word to the sentence and then the passage in oral reading fluency as well as both the direct and indirect importance of word-level oral reading fluency in reading comprehension. Only the indirect effect from word-level oral reading fluency to reading comprehension through passage-level oral reading fluency was significant. Our findings suggest that sentence-level oral reading fluency is the crucial component to reading comprehension in Chinese. Additionally, recognition of the potential value of unique features, such as syntactic awareness and word segment accuracy, that happen at the sentence level should be integrated into instructional activities for reading.

Difficult catheterization and previous urethral obstruction are associated with lower urinary tract tears in cats with urethral obstruction.

To retrospectively assess the hospital prevalence and risk factors associated with iatrogenic lower urinary tear in cats with urethral obstruction (UO). 15 client-owned cats diagnosed with concurrent UO and lower urinary tears and year-matched control population of 45 cats diagnosed with UO. University teaching hospital records were reviewed for cats presenting with UO between January 2010 and December 2022. Signalment, anatomic location of tear, experience of the individual passing the urinary catheter, difficulty level of catheter passage, history of previous UO, blood work parameters on presentation, presence of visible grit in urine, and survival to discharge were recorded. In addition, prevalence of lower urinary tears in cats presenting with UO was calculated. The prevalence of lower urinary tears was 0.92% in UO cats. Cats with lower urinary tears were significantly less likely to survive to discharge and had a longer period of hospitalization than cats without tears. In addition, cats with tears were more likely to have a history of previous UO and had more difficult urinary catheter passage than cats in the control group. Cats with tears also had a higher Hct than the control UO cats. Cats that develop lower urinary tears are more likely to have a history of previous UO and difficult catheter passage. This group of cats is also more likely to have a longer hospitalization period and lower survival to discharge rates.

Development and characterization of brain cell line from Trachinotus blochii and its application in virological and gene expression studies.

A permanent cell line, SPB (Snubnose pompano brain) was established from Trachinotus blochii by the explant culture method. It has been sub-cultured more than 75 passages and showed optimal growth at 28°C using L-15 medium supplemented with 15% to 20% FBS. The SPB cells were cryopreserved at different passage levels for various applications. SPB cells were composed of fibroblastic and epithelial-like cells. The SPB cells were tested for mycoplasma contamination which was found to be negative. The origin of the SPB cell line from T. blochii was confirmed by amplification of the mitochondrial cytochrome oxidase I (COI) gene. The transfection efficiency of SPB cell line is 15% assessed by expression of green fluorescent protein using pEGFP-N1 plasmid. In addition, two CMV promotor plasmids pFNCPE42-DNA and pcDNAVP28 were transfected to SPB cells and it shows high expression levels of FNCP of fish nodavirus and VP28 protein of white spot syndrome virus by immunostaining. The SPB cells showed susceptibility to SJNNV and the infection was confirmed by RT-PCR, Western blot, ELISA, TCID50 and RT-qPCR. Experimental infection was carried out in T. blochii using SJNNV propagated in SPB cell line and found 100% mortality with clinical signs. The infection was confirmed by RT-PCR. The SPB cell line can be used for propagation of fish viral pathogens and production of the recombinant proteins.

The first lumpy skin disease outbreak in cattle in Kazakhstan

Lumpy skin disease (LSD) is an emerging trans-boundary viral disease of cattle originating from the African continent. Here, we describe the first LSD outbreak reported in the Republic of Kazakhstan in July 2016, as well as associated clinical manifestations of the disease, diagnostic methods, and control measures taken to combat further spread of the pathogen. To determine the cause of the disease, samples were taken from sick and dead animals, as well as from insects and ticks. LSDV DNA was detected by PCR in all samples from dead animals and ticks (Dermacentor marginatus and Hyalomma asiaticum), in 14.29% of samples from horseflies (Tabanus bromius), and in one of the samples from two Stomoxys calcitrans flies. The reproductive LSD virus was isolated from organs of dead cattle and insects in the culture of LT and MDBK cells. The virus accumulated in cell cultures of LT and MDBK at the level of the third passage with titers in the range of 5.5–5.75 log 10 TCID50/cm3.During the outbreak, the number of affected cattle within an epidemiological unit reached 459 cattle out of 3557 registered susceptible cattle, with 12.90% morbidity and 0.96% mortality. This manuscript presents the epidemiological situation; the diagnosis; the control measures, including mass vaccination; and the stamping out campaign.

The Influence of Passage Cohesion on Cloze Test Item Difficulty

The role of context in cloze tests has long been seen as both a benefit as well as a complication in their usefulness as a measure of second language comprehension (Brown, 2013). Passage cohesion, in particular, would seem to have a relevant and important effect on the degree to which cloze items function and the interpretability of performances (Brown, 1983; Dastjerdi &amp; Talebinezhad, 2006; Oller &amp; Jonz, 1994). With recent evidence showing that cloze items can require examinees to access information at both the sentence and passage level (Trace, 2020), it’s worthwhile to now look back and examine the relationship between aspects of passage cohesion—referential cohesion, semantic overlap, and incidence of conjunctives—and item difficulty by classification. The current study draws upon a large pool of cloze test passages and items (k = 377) originally used by Brown (1993) along with automated text analysis of cohesion (Coh-Metrix, McNamara et al., 2014) to examine the impact of passage cohesion on item function. Correlations, factor analysis, and linear regression point to clear though minimal differences for both sentential and intersentential items as they relate to aspects of passage cohesion, the results of which may inform future test design and interpretation of cloze performance.

Comparative Analysis of MEM, RPMI 1640 Media on Rabies Virus Propagation in Vero Cells and Virus Quantification by FAT

Rabies disease can be preventable through vaccination is the only way for effective control and the vaccine is administered as post exposure prophylaxis method (PEP) along with rabies immunoglobulin. For the preparation of the vaccine, the high titre rabies virus has to be propagated in suitable host system like Vero cells. In this study, the virus was propagated in tissue culture flask in two different tissue culture media namely MEM and RPMI-1640, keeping Vero cell line as the host system. This preliminary study is to find the media that yields a better viral titre as high, the influence of harvesting intervals on viral titre for the reason that the vaccine yield is directly proportional to the viral titre during the virus propagation stage and leads to cost effective vaccine. The Vero cell was revived from passage 154 and the part of cells were subjected for adaptation in RPMI-1640 media with gradual media replacement. The passaging of RPMI 1640 media cells were continued until it reaches the equal cell count of MEM (p-158). The confluent monolayer of Vero cells was maintained in the same passage level with appropriate media. The cell count of MEM media, RPMI-1640 were 10.28 x 106 and 10.35 x 106 respectively in 25cm2 tissue culture flasks. The Vero cells were sub-cultured in 175 cm2 tissue culture flasks infected with 0.2 MOI (Multiplicity of Infection) of virus and the viral titration was estimated through FAT test (Fluorescent Antibody Test). The highest viral titre obtained from RPMI-1640 media batch (10-6.125/ml) and MEM media batch (10-6.25 /ml). The two days interval viral harvests shows (Batch 1 &amp; 3) the viral titre log ranged between 10-4.375 to 10-5.875 and three days interval harvests (Batch 2 &amp; 4) the viral titre log ranged between 10-3.750 to 10-6.250 per mL.

Isolation and Identification of Caprine Arthritis Encephalitis Virus from Animals in the Republic of Mordovia.

This article presents the results of virological and genetic studies of an isolate of caprine arthritis encephalitis (CAE) virus from the republic of Mordovia, Russian Federation. The isolate was found during monitoring studies of goat blood samples for the viral genome, and the presence of antibodies to lentiviruses was detected. According to the recommendation of the OIE, the positive result of PCR was confirmed with nucleotide sequencing. It was found that the obtained nucleotide sequence is identical to the genome of small ruminant lentiviruses presented in the GenBank database. Phylogenetic analysis showed that the isolate "Mordovia-2018" was included in the same cluster with an isolate from the Tver region of the Russian Federation detected in 2008. The sequence of the fragment of the env-gene of the isolate from the republic of Mordovia is available in GenBank under the number MN186380.1. To isolate the virus, a fraction of peripheral blood monocyte cells from the animal's blood was added to a monolayer of lamb synovial membrane cell culture, and ten passages were carried out. The first manifestations of the cytopathic effect were observed after the third passage on the eighth day of cultivation in the form of single large cells of irregular shape with 5-7 nuclei. At the seventh passage, multiple syncytium with 7-12 nuclei were observed. At subsequent passage levels, the formation of syncytium containing more than 10-14 nuclei was observed.

A Corpus-based Analysis of the Linguistic Complexity Levels of the Reading Passages in Three Asian EFL Countries’ College Entrance English Exams

This study investigates the linguistic complexity levels of the reading passages of the foreign language (English) domain of the College Scholastic Aptitude Test (CSAT) in Korea and compares them with those in two other Asian EFL countries. For the study, the reading passages of the Korean, Chinese, and Japanese college entrance English exams from the 2018 to 2020 academic year were collected and analyzed based on the length, propositional content and organizational characteristics by using a variety of corpus linguistic tools. The Kruskal-Wallis test was administered in order to see if there was a statistically significant difference among the tests. Also, when between-group differences were detected, the Mann-Whitney U post hoc test was conducted to further look into significant differences between the CSAT and the other tests. Overall findings suggest that the reading passages of the CSAT English exam maintained the highest difficulty level among the three Asian EFL countries’ college entrance English exams, with cognitively demanding academically-oriented items, highly advanced vocabulary, complex syntactic structure and low readability. Based on the findings, this research is expected to shed light on the intention of incorporating criterion-referenced English assessment into the CSAT as well as provide pedagogical implications for test developers and education policy makers in Korea.

Development of cell culture based peste des petits ruminants (PPR) virus vaccine candidate from Bangladeshi isolates

This study was conducted to develop a cell culture based PPR virus vaccine candidate using recent Bangladeshi strain of peste des petits ruminant’s (PPR) virus. PPR virus was isolated from field outbreaks, confirmed by RT-PCR and used as viral inoculum for serial passaging in Vero cells for adaptation and attenuation. 60th serial passage had completed and RT-PCR and real time RT-PCR were done in every 5 passages for confirmation of PPR virus in tissue culture fluid (TCF). To assess the adaptation and attenuation cytopathology, virus titration, sequencing of both F and N genes and live animal experimentation were done. Different cellular alterations produced by PPR virus in infected Vero cells including syncytia formation, development of both intranuclear and intra cytoplasmic inclusion bodies and finally cell degradation are the indications of adaptation. The virus titre was found 2.5, 3.31, 3.55, 4.44, 4.71 and 6.5 Log10 TCID50/ml at 10th, 20th, 30th, 40th, 50th and 60th passages level respectively. In F gene sequence analysis it has been observed that few nucleotide (nt) and mino acid (aa) has been substituted as the effects of serial passaging of PPR virus in Vero cells. TCF at 60th passage level was found effective to produced protective antibody (Ab) titre in live animal experimentation. It is concluded that serially passaged and Vero cells adapted PPR virus TCF could be used as a vaccine candidate for further use to develop a potent & effective vaccine against PPR diseases.

Adaptation of the rabies virus of the production strain "sheep" GNKI to the transplanted cultures of NGUK-1 and ВНК-21/13 CELLS

The selection of the optimal line of the transplanted cell culture and the adaptation of the rabies virus to it makes it possible to exclude the use of laboratory animals, fully control the process of obtaining viruscontaining material with high infectious activity in large quantities and with a faster and shorter production cycle. The adaptive potential of various strains of rabies virus varies significantly, in this regard, the aim of the study was to study the sensitivity of the production strain of the rabies virus "Sheep" GNKI to the transplanted cultures of NGUK-1 and ВНК-21/13 cells. The adaptation of the rabies virus to the transplanted cultures of NGUK-1 and VNK-21/13 cells was carried out by sequential passivation. The titer of the virus was calculated by the number of fluorescent foci, the concentration of rabies virus antigen was determined by the ELISA method, the pathogenicity of the virus at the level of 11 passages on different cell lines – on white mice.It was found that the transplanted ВНК-21/13 cell line provided a faster adaptation of the virus and the achievement of maximum titers within 36-48 hours, whereas the NGUK-1 line maintained relatively slow replication and ensured the achievement of maximum titers after 96-120 hours. The optimal multiplicity of infection with NGUK-1 and ВНК-21/13 was 0.1 KKID50/cell, while the titers of the "Sheep" virus strains were 4.11±0.26 and 6.17±0.49 lg KKID50/cm3, respectively. Virus replication in ВНК-21/13 cells was characterized by greater intensity: the antigenic titer of the virus at all passage levels was 1.5-2 times (p&lt;0.05) higher than that in NGUK-1 cells. The positive dynamics of the accumulation of the viral titer persisted until passage 8-9, after which this indicator remained stable until passage 11 inclusive. The results of the assessment of the pathogenicity of the rabies virus at the level of passage 11 on different cell lines showed that the virus adapted to both NGUK-1 and ВНК-21/13 has not lost pathogenicity for white mice. Thus, it was found that the transplanted ВНК-21/13 cell line significantly exceeds the NGUK-1 cell line in terms of replicative capabilities and can be used to develop viral raw materials for the production of diagnostic tools and specific prevention.

A Spontaneously Occurring African Swine Fever Virus with 11 Gene Deletions Partially Protects Pigs Challenged with the Parental Strain.

African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 105 TCID50 of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development.

Свадебная обрядовая одежда как феномен духовной культуры в южнорусском селе XIX–XX вв.: семиотический аспект

Traditional clothing was an integral attribute of the spiritual and cultural space of a Russian village until the 20th century. In connection with the destruction of traditional and the subsequent resemanticisation of folk clothing in the 19th – 21th centuries, there was a need to clarify the main spiritual and cultural content of a traditional costume. Wedding ceremonial clothing was chosen as the object of research due to its high semiotic status and good preservation, which provides extensive research field. The empirical basis of the work consists of ethnographic descriptions of the studied origin regions of wedding suit, private collections, museum funds and the photo archive of the author, formed as a result of expeditions 2018–2019. The article considers the totality of all wedding sets of bride's clothes as a unified semiotic system. The context of this phenomenon is an entire syntagmatic sequence of wedding ceremonies, starting from matchmaking and ending with the last day of a wedding. The wedding in this case is considered as a rite of passage of a life cycle, the main leitmotif of which is the process of “dying-death-rebirth”. Special costume sets mark all stages of the passage (preliminary, liminary, postliminary): “sad” and “grief” clothes are gradually replaced by “kind”, “cheerful”, white and dark shades are replaced by bright, often in red, and, in general, the semantics of death is replaced by the semantics of life. The study also shows that wedding cultures of different villages represent different semiotic systems that function on the basis of a single principle of passage, but some of its aspects and symbolization may be different. At the same time, a complex relationship between the social and spiritual levels of passage is realized, the markers of which may be different characteristics of the same costume complex. In general, the semiotics of wedding ceremonial clothing testifies to the unity of the spiritual and social spheres of a Russian village in the 19th – 20th centuries.

A Review of the Establishment of the Seed Lot System in the Production of Biological Products and Its Importance.

Today, due to the importance of diseases controlled by vaccination, the production of biological products is of great importance in ensuring public health, so producing high-quality biological products plays an important role in maintaining public health. One of the most important principles of manufacturing high-quality and efficient biological products is using suitable seeds based on standard principles. To produce a suitable seed for continuous use for mass production of biological products, the seed must be defined according to certain principles and foundations. These principles are formed in the form of a seed lot system. In this review article, all the requirements for the establishment of a seed to produce a biological product include general seed information, including basic and seed information and microorganisms, seed-specific information including seed passage levels, propagation method, seed storage conditions, coding and labeling, identification information and design of suitable laboratories for passage, seed propagation, and storage, determination of seed characteristics including all necessary tests to determine seed identity, purity, potency, efficacy, stability, safety and also all the necessary information for documenting and storing seeds has been studied. Also, this study discusses the method of preparing all the necessary information for establishing a seed lot system, especially determining the characteristics of seeds. Based on the study's results, a complete and comprehensive seed lot system has been formed that can be used to prepare, propagate, passage, and store seeds used in the production of biological products.

Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio).

The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio. A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28°C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48h post-transfection. The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.