New strategies for germ cell cryopreservation: Cryoinjury modulation / 대한생식의학회지

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

Influence of a new polishing system on changes in gloss and surface roughness of resin composites after polishing and brushing.

This study aimed to compare the change of surface roughness (Ra) and gloss units (GU) of five dental composites (Filtek Z250, Filtek Z350XT, Metafil CX, Ceram X one, and Venus Diamond) polished with three systems (Sof-Lex XT, Enhance/Pogo, and Sof-Lex Diamond) before/after simulated brushings and to determine the amount of time required to achieve maximum gloss. Ninety rectangular specimens (n=18 per composite) were prepared. Six specimens of each composite were assigned to one of the polishing systems. The Ra and GU of each specimen were measured after each polishing step. Five polished specimens per composite were brushed with a toothbrush machine, and the Ra and GU values were determined. Filtek Z350XT exhibited the most stable and lowest Ra during the brushing cycles regardless of polishing system. When using the Sof-Lex Diamond and Enhance/Pogo systems, the highest gloss and the smoothest surfaces were achieved after polishing and brushing.

Direct modification of spermatogonial stem cells using lentivirus vectors in vivo leads to efficient generation of transgenic rats / 亚洲男科学杂志(英文版)

Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.

Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes

Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 ℃. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.

Characteristics of Patients Injured in Road Traffic Accidents According to the New Injury Severity Score

OBJECTIVE: To investigate the clinical characteristics of patients involved in road traffic accidents according to the New Injury Severity Score (NISS). METHODS: In this study, medical records of 1,048 patients admitted at three hospitals located in different regions between January and December 2014 were retrospectively reviewed. Only patients who received inpatient treatments covered by automobile insurance during the period were included. Accidents were classified as pedestrian, driver, passenger, motorcycle, or bicycle; and the severity of injury was assessed by the NISS. RESULTS: The proportion of pedestrian traffic accident (TA) was the highest, followed by driver, passenger, motorcycle and bicycle TA. The mean NISS was significantly higher in pedestrian and motorcycle TAs and lower in passenger TA. Analysis of differences in mean hospital length of stay (HLS) according to NISS injury severity revealed 4.97±4.86 days in the minor injury group, 8.91±5.93 days in the moderate injury group, 15.46±11.16 days in the serious injury group, 24.73±17.03 days in the severe injury group, and 30.86±34.03 days in the critical injury group (p<0.05). CONCLUSION: The study results indicated that higher NISS correlated to longer HLS, fewer home discharges, and increasing mortality. Specialized hospitals for TA patient rehabilitation are necessary to reduce disabilities in TA patients.

Extraction sockets: erratic healing impeding factors.

AIM: The aim of this exploratory study was to analyse prevalence of extraction sockets showing erratic healing and evaluate factors potentially impeding healing. METHODS: Erratic healing was defined as extraction sites showing clinical observations of fibrous scar tissue occupying the extraction site rather than bone following 12 or more weeks of healing. Computerized tomography was used to evaluate characteristics and calculate Hounsfield unit scores for sites showing erratic healing. RESULTS: A total of 1226 dental records from Seoul National University Dental Hospital archives including patients subject to extractions prior to implant placement were evaluated. Seventy subjects (5.71%) and 97 sites (4.24%) exhibited erratic extraction socket healing. Maxillary incisor/canine sites showed the lowest (0.47%), whereas mandibular molar sites the highest (5.41%) occurrence. In the multivariable analysis, erratic healing was more likely to occur in subjects <60 years old (OR = 2.23, 95%CI = 1.26-3.94), subjects with hypertension (OR = 2.37, 95%CI = 1.24-4.55), in molar sites (OR = 4.91, 95%CI = 1.41-17.07), and following single tooth extractions (OR = 2.98, 95%CI = 1.36-6.53). Computerized tomography showed the highest incidence of bone loss for the buccal wall (49.3%). CONCLUSION: Erratic extraction socket healing appears a not uncommon sequel and local factors seem to be major contributors to its occurrence.

Sequence Generation and Genotyping of 15 Autosomal STR Markers Using Next Generation Sequencing / 대한법의학회지

Recently, next generation sequencing (NGS) has received attention as the ultimate genotyping method to overcome the limitations of capillary electrophoresis (CE)-based short tandem repeat (STR) analysis, such as the limited number of STR loci that can be measured simultaneously using fluorescent-labeled primers and the maximum size of STR amplicons. In this study, we analyzed 15 autosomal STR markers via the NGS method and evaluated their effectiveness in STR analysis. Using male and female standard DNA as single-sources and their 1:1 mixture, we sequentially generated sample amplicons by the multiplex polymerase chain reaction (PCR) method, constructed DNA libraries by ligation of adapters with a multiplex identifier (MID), and sequenced DNA using the Roche GS Junior Platform. Sequencing data for each sample were analyzed via alignment with pre-built reference sequences. Most STR alleles could be determined by applying a coverage threshold of 20% for the two single-sources and 10% for the 1:1 mixture. The structure of the STR in each allele was accurately determined by examining the sequences of the target STR region. The mixture ratio of the mixed sample was estimated by analyzing the coverage ratios between assigned alleles at each locus and the reference/variant ratios from the observed sequence variations. In conclusion, the experimental method used in this study allowed the successful generation of NGS data. In addition, the NGS data analysis protocol enables accurate STR allele call and repeat structure determination at each locus. Therefore, this approach using the NGS system will be helpful to interpret and analysis the STR profiles from singe-source and even mixed samples in forensic investigation.