The Effect of Prohibitins on Mitochondrial Function during Octopus tankahkeei Spermiogenesis.

Mitochondria are essential for spermiogenesis. Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins that act as scaffolds in the inner mitochondrial membrane. In this study, we analyzed the molecular structure and dynamic expression characteristics of Ot-PHBs, observed the colocalization of Ot-PHB1 with mitochondria and polyubiquitin, and studied the effect of phb1 knockdown on mitochondrial DNA (mtDNA) content, reactive oxygen species (ROS) levels, and apoptosis-related gene expression in spermatids. Our aim was to explore the effect of Ot-PHBs on mitochondrial function during the spermiogenesis of Octopus tankahkeei (O. tankahkeei), an economically important species in China. The predicted Ot-PHB1/PHB2 proteins contained an N-terminal transmembrane, a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin domain), and a C-terminal coiled-coil domain. Ot-phb1/phb2 mRNA were widely expressed in the different tissues, with elevated expression in the testis. Further, Ot-PHB1 and Ot-PHB2 were highly colocalized, suggesting that they may function primarily as an Ot-PHB compiex in O. tankahkeei. Ot-PHB1 proteins were mainly expressed and localized in mitochondria during spermiogenesis, implying that their function may be localized to the mitochondria. In addition, Ot-PHB1 was colocalized with polyubiquitin during spermiogenesis, suggesting that it may be a polyubiquitin substrate that regulates mitochondrial ubiquitination during spermiogenesis to ensure mitochondrial quality. To further investigate the effect of Ot-PHBs on mitochondrial function, we knocked down Ot-phb1 and observed a decrease in mtDNA content, along with increases in ROS levels and the expressions of mitochondria-induced apoptosis-related genes bax, bcl2, and caspase-3 mRNA. These findings indicate that PHBs might influence mitochondrial function by maintaining mtDNA content and stabilizing ROS levels; in addition, PHBs might affect spermatocyte survival by regulating mitochondria-induced apoptosis during spermiogenesis in O. tankahkeei.

Expression Dynamics Indicate Potential Roles of KIF17 for Nuclear Reshaping and Tail Formation during Spermiogenesis in Phascolosoma esculenta.

Kinesin family member17 (KIF17), a homologous dimer of the kinesin-2 protein family, has important microtubule-dependent and -independent roles in spermiogenesis. Little is known about KIF17 in the mollusk, Phascolosoma esculenta, a newly developed mariculture species in China. Here, we cloned the open reading frame of Pe-kif17 and its related gene, Pe-act, and performed bioinformatics analysis on both. Pe-KIF17 and Pe-ACT are structurally conserved, indicating that they may be functionally conserved. The expression pattern of kif17/act mRNA performed during spermiogenesis revealed their expression in diverse tissues, with the highest expression level in the coelomic fluid of P. esculenta. The expressions of Pe-kif17 and Pe-act mRNA were relatively high during the breeding season (July-September), suggesting that Pe-KIF17/ACT may be involved in spermatogenesis, particularly during spermiogenesis. Further analysis of Pe-kif17 mRNA via fluorescence in situ hybridization revealed the continuous expression of this mRNA during spermiogenesis, suggesting potential functions in this process. Immunofluorescence showed that Pe-KIF17 co-localized with α-tubulin and migrated from the perinuclear cytoplasm to one side of the spermatid, forming the sperm tail. Pe-KIF17 and Pe-ACT also colocalized. KIF17 may participate in spermiogenesis of P. esculenta, particularly in nuclear reshaping and tail formation by interacting with microtubule structures similar to the manchette. Moreover, Pe-KIF17 with Pe-ACT is also involved in nuclear reshaping and tail formation in the absence of microtubules. This study provides evidence for the role of KIF17 during spermiogenesis and provides theoretical data for studies of the reproductive biology of P. esculenta. These findings are important for spermatogenesis in mollusks.

Application of a Caries Treatment Difficulty Assessment System in Dental Caries Management.

Dental caries is one of the most common chronic diseases caused by progressive bacteria, affecting all age groups. Today, restorative fillings are widely used for dental caries treatment, but the restorative treatment has a high failure rate. Meanwhile, many researchers have discovered the differences of caries risk among populations by using the caries risk assessment and put forward a new standpoint that caries should be treated individually. Therefore, our research group established a Dental Caries Treatment Difficulty Assessment system in a previous study. This time, we combined the caries risk assessment with the caries treatment difficulty assessment, then used Python to design a Dental Caries Management Software. The purpose of this case report is to present a case applying this software in dental caries management and other data collected in Chengdu, China, with this software on the assessment of caries treatment difficulty. Patients with personalized assessment and management can achieve good treatment results, including reducing the risk and treatment difficulty of dental caries. At the same time, other cases show that the software has good application potential in individual management and group information collection. These cases indicate that the software enables dentists to carry out both the risk assessments and the treatment difficulty assessment of patients, and it has the potential as a tool for epidemiological investigation. It also enables dentists and patients to have a basic understanding of the dental health status of patients and create personalized dental caries treatment, so as to achieve the goal of controlling the progression of dental caries and rebuilding the structure and restoring the function of teeth.

Characterization of Mitochondrial Prohibitin in Opsariichthys bidens and Its Potential Functions in Spermatogenesis.

Spermatogenesis is the intricate and coordinated process by which spermatogonia develop into haploid differentiated spermatozoa. Mitochondria are essential for spermatogenesis, and prohibitin (PHB) is closely associated with mitochondrial structure and function during spermatogenesis. Although PHB has been implicated in spermatogenesis in some taxa, its roles in Opsariichthys bidens have not been determined. In this study, the expression patterns and potential functions of PHB in spermatogenesis in O. bidens were characterized using histological microscopic observations, PCR cloning, real-time quantitative PCR (qPCR), Western blotting (WB) and immunofluorescence (IF). The full-length cDNA of Ob-phb was 1500 bp encoding 271 amino acids. A sequence alignment demonstrated that the PHB protein is conserved among different animals. qPCR revealed that phb mRNA is widely distributed in O. bidens and highly expressed in the testes at stages IV and V. WB revealed that Ob-PHB is located in the mitochondria of testes. IF revealed the colocalization of PHB signals and mitochondria. Signals were detected around nuclei in spermatogonia and spermatocytes, gradually moving to the tail region during spermiogenesis, and finally aggregating in the midpiece. These results indicate that Ob-PHB was expressed in the mitochondria during spermatogenesis. In addition, this study proposed Ob-PHB may participate in the degradation of mitochondria and cell differentiation during spermatogenesis.

The potential function of KIF17 in large yellow croaker (Larimichthys crocea) spermatid remodeling: molecular characterization and expression pattern during spermiogenesis.

KIF17, which belongs to the kinesin-2 protein family, plays an indispensable role in mammalian spermiogenesis. However, the role of KIF17 in fish spermatid remodeling during spermiogenesis remains poorly understood. Therefore, we aimed to study the role of KIF17 in spermatid remodeling during Larimichthys crocea (L. crocea) spermiogenesis. The kif17 cDNA sequence, 3247 bp in length, was cloned from L. crocea testis, which consisted of a 347-bp 5'-untranslated region (UTR), 413-bp 3' -UTR, and 2487-bp open reading frame. Bioinformatic analyses revealed that KIF17 obtained from L. crocea (Lc-KIF17) exhibited a high sequence identity compared with those from other teleosts and possessed the structural features of other kinesin-2 proteins. Based on structural similarity, we speculate that the role of Lc-KIF17 may be similar to that of KIF17 in other animals. Lc-kif17 mRNA was diffusely expressed in L. crocea tissues and was highly expressed in the testis, especially at stage IV testicular development. Immunofluorescence analysis revealed that Lc-KIF17 signals colocalized with ß-tubulin signals and migrated from the perinuclear cytoplasm to the side of the nucleus where the tail forms during spermiogenesis. These findings revealed that KIF17 may be involved in L. crocea spermiogenesis. In particular, KIF17 may participate in spermatid remodeling by interacting with perinuclear microtubules during L. crocea spermiogenesis. Collectively, this study contributes to an improved understanding of the mechanism underlying L. crocea spermiogenesis and provides a basis for further research on L. crocea reproduction and development.

Genetics Responses to Hypoxia and Reoxygenation Stress in Larimichthys crocea Revealed via Transcriptome Analysis and Weighted Gene Co-Expression Network.

The large yellow croaker (Larimichthys crocea) is an important marine economic fish in China; however, its intolerance to hypoxia causes widespread mortality. To understand the molecular mechanisms underlying hypoxia tolerance in L. crocea, the transcriptome gene expression profiling of three different tissues (blood, gills, and liver) of L. crocea exposed to hypoxia and reoxygenation stress were performed. In parallel, the gene relationships were investigated based on weighted gene co-expression network analysis (WGCNA). Accordingly, the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that several pathways (e.g., energy metabolism, signal transduction, oxygen transport, and osmotic regulation) may be involved in the response of L. crocea to hypoxia and reoxygenation stress. In addition, also, four key modules (darkorange, magenta, saddlebrown, and darkolivegreen) that were highly relevant to the samples were identified by WGCNA. Furthermore, some hub genes within the association module, including RPS16, EDRF1, KCNK5, SNAT2, PFKL, GSK-3ß, and PIK3CD, were found. This is the first study to report the co-expression patterns of a gene network after hypoxia stress in marine fish. The results provide new clues for further research on the molecular mechanisms underlying hypoxia tolerance in L. crocea.

Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker ( Larimichthys crocea) under environmental hypoxia.

Oxygen is an essential molecule for animal respiration, growth, and survival. Unlike in terrestrial environments, contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments, thus impacting aquatic animal survival. However, the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown. Here, we used large yellow croaker ( Larimichthys crocea) and large yellow croaker fry (LYCF) cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis, and apoptosis. We confirmed that hypoxia induced the expression of Hif-1α, Hsf1, and Hsp70 in vivo and in vitro. Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress. Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential, enhancing Bcl-2 mRNA and protein expression, inhibiting Bax and caspase3 mRNA expression, and suppressing caspase-3 and caspase-9 activation. Hsp70 suppressed caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription. Hsf1 enhanced Hsp70 mRNA transcription in a similar manner. In summary, the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L. crocea under hypoxic stress.

Scavenging reactive oxygen species is a potential strategy to protect Larimichthys crocea against environmental hypoxia by mitigating oxidative stress.

The large yellow croaker (Larimichthys crocea), which is an economically important mariculture fish in China, is often exposed to environmental hypoxia. Reactive oxygen species (ROS) homeostasis is essential for the maintenance of normal physiological conditions in an organism. Direct evidence that environmental hypoxia leads to ROS overproduction is scarce in marine fish. Furthermore, the sources of ROS overproduction in marine fish under hypoxic stress are poorly known. In this study, we investigated the effects of hypoxia on redox homeostasis in L. crocea and the impact of impaired redox homeostasis on fish. We first confirmed that hypoxia drove ROS production mainly via the mitochondrial electron transport chain and NADPH oxidase complex pathways in L. crocea and its cell line (large yellow croaker fry (LYCF) cells). We subsequently detected a marked increase in the antioxidant systems of the fish. However, imbalance between the pro-oxidation and antioxidation systems ultimately led to excessive ROS and oxidative stress. Cell viability showed a remarkable decrease while oxidative indicators, such as malondialdehyde, protein carbonylation, and 8-hydroxy-2 deoxyguanosine, showed a significant increase after hypoxia, accompanied by tissue damage. N-acetylcysteine (NAC) reduced ROS levels, alleviated oxidative damage, and improved cell viability in vitro. Appropriate uptake of ROS scavengers (e.g., NAC and elamipretide Szeto-Schiller-31) and inhibitors (e.g., apocynin, diphenylene iodonium, and 5-hydroxydecanoate) may be effective at overcoming hypoxic toxicity. Our findings highlight previously unstudied strategies of hypoxic toxicity resistance in marine fish.

KIFC1 functions in nuclear reshaping and midpiece formation during the spermatogenesis of small yellow croaker Larimichthys polyactis.

The C-terminal kinesin motor protein (KIFC1) has essential functions in spermatogenesis. To evaluate molecular mechanisms of KIFC1 during teleost fish spermatogenesis, there was cloning and sequencing the kifc1 cDNA in the testis of Larimichthys polyactis. Quantitative PCR results indicated there were Lp-kifc1 mRNA transcripts in the testes. Results from conducting fluorescence in situ hybridization and immunofluorescence procedures indicated there were trends in relative abundance changes in Lp-kifc1 mRNA transcripts that were associated with abundance of Lp-KIFC1 protein during spermatogenesis. The Lp-KIFC1 protein was detected at all stages of spermatogenesis. There was minimal Lp-KIFC1 in the cytoplasm of spermatogonia, with content being greater and concentrated in the perinuclear region in spermatocytes and during early/mid-stages of development of spermatids. There were large abundances of Lp-KIFC1 in spermatids at the mid-developmental stage. In late-developing spermatids, Lp-KIFC1 content was less and concentrated in the bottom of the nucleus, where the midpiece formed. There was a small Lp-KIFC1 in the midpiece of mature sperm. These findings indicate Lp-KIFC1 may have functions in L. polyactis spermatogenesis. Results from conducting immunofluorescence procedures indicated Lp-KIFC1 was co-localized microtubules and mitochondria throughout spermatogenesis. There were large abundances of Lp-KIFC1 and tubulin in spermatids during the mid-developmental stage, when there is a decrease in size and reshaping of the nucleus. During midpiece formation, there was co-localization of the Lp-KIFC1 and mitochondria in the spermatid perinuclear region to the midpiece. These findings indicate Lp-KIFC1 is involved in nuclear reshaping and midpiece formation during spermatogenesis in L. polyactis.

Expression and potential functions of KIF3A/3B to promote nuclear reshaping and tail formation during Larimichthys polyactis spermiogenesis.

KIF3A and KIF3B are homologous motor subunits of the Kinesin II protein family. KIF3A, KIF3B, and KAP3 form a heterotrimeric complex and play a significant role in spermatogenesis. Here, we first cloned full-length kif3a/3b cDNAs from Larimichthys polyactis. Lp-kif3a/3b are highly related to their homologs in other animals. The proteins are composed of three domains, an N-terminal head domain, a central stalk domain, and a C-terminus tail domain. Lp-kif3a/3b mRNAs were found to be ubiquitously expressed in the examined tissues, with high expression in the testis. Fluorescence in situ hybridization (FISH) was used to analyze the expression of Lp-kif3a/3b mRNAs during spermiogenesis. The results showed that Lp-kif3a/3b mRNAs had similar expression pattern and were continuously expressed during spermiogenesis. From middle spermatid to mature sperm, Lp-kif3a/3b mRNAs gradually localized to the side of the spermatid where the midpiece and tail form. In addition, we used immunofluorescence (IF) to observe that Lp-KIF3A protein co-localizes with tubulin during spermiogenesis. In early spermatid, Lp-KIF3A protein and microtubule signals were randomly distributed in the cytoplasm. In middle spermatid, however, the protein was detected primarily around the nucleus. In late spermatid, the protein migrated primarily to one side of the nucleus where the tail forms. In mature sperm, Lp-KIF3A and microtubules accumulated in the midpiece. Moreover, Lp-KIF3A co-localized with the mitochondria. In mature sperm, Lp-KIF3A and mitochondria were present in the midpiece. Therefore, Lp-KIF3A/KIF3B may be involved in spermiogenesis in L. polyactis, particularly during nuclear reshaping and tail formation.

Automatic segmentation of cardiac substructures from noncontrast CT images: accurate enough for dosimetric analysis?

PURPOSE: We evaluated the feasibility of using an automatic segmentation tool to delineate cardiac substructures from noncontrast computed tomography (CT) images for cardiac dosimetry and toxicity analyses for patients with nonsmall cell lung cancer (NSCLC) after radiotherapy. MATERIAL AND METHODS: We used an in-house developed multi-atlas segmentation tool to delineate 11cardiac substructures, including the whole heart, four heart chambers, and six greater vessels, automatically from the averaged 4D-CT planning images of 49 patients with NSCLC. Two experienced radiation oncologists edited the auto-segmented contours. Times for automatic segmentation and modification were recorded. The modified contours were compared with the auto-segmented contours in terms of Dice similarity coefficient (DSC) and mean surface distance (MSD) to evaluate the extent of modification. Differences in dose-volume histogram (DVH) characteristics were also evaluated for the modified versus auto-segmented contours. RESULTS: The mean automatic segmentation time for all 11 structures was 7-9 min. For the 49 patients, the mean DSC values (±SD) ranged from .73 ± .08 to .95 ± .04, and the mean MSD values ranged from 1.3 ± .6 mm to 2.9 ± 5.1 mm. Overall, the modifications were small; the largest modifications were in the pulmonary vein and the inferior vena cava. The heart V30 (volume receiving dose ≥30 Gy) and the mean dose to the whole heart and the four heart chambers were not different for the modified versus the auto-segmented contours based on the statistically significant condition of p < .05. Also, the maximum dose to the great vessels was no different except for the pulmonary vein. CONCLUSIONS: Automatic segmentation of cardiac substructures did not require substantial modifications. Dosimetric evaluation showed no significant difference between the auto-segmented and modified contours for most structures, which suggests that the auto-segmented contours can be used to study cardiac dose-responses in clinical practice.

Technical Note: Solving the "Chinese postman problem" for effective contour deformation.

PURPOSE: To develop a practical approach for accurate contour deformation when deformable image registration (DIR) is used for atlas-based segmentation or contour propagation in image-guided radiotherapy. METHODS: We developed a contour deformation approach based on 3D mesh operations. The 2D contours represented by a series of points in each slice were first converted to a 3D triangular mesh, which was deformed by the deformation vectors resulting from DIR. A set of parallel 2D planes then cut through the deformed 3D mesh, generating unordered points and line segments, to be reorganized into a set of 2D contour points. The reorganization problem was equivalent to solving the "Chinese postman problem" (CPP) by traversing a graph built from the unordered points with the least cost. Alternatively, deformation could be applied to a binary image converted from the original contours. The deformed binary image was then converted back into contours at the CT slice locations. We validated the mesh-based contour deformation approach using lung and heart contours from 10 patients with thoracic cancer. RESULTS: DIR could change the 3D mesh considerably, complicating 2D contour representations after deformation. CPP could effectively reorganize the points in 2D planes regardless of how complicated the 2D contours were. Among the 10 patients, the Dice similarity coefficient between the mesh-based contour and binary image-based contour was 97.6% ± 0.3% for lung and 97.5% ± 0.7% for heart, and the Hausdoroff distance between them was 19.8 ± 5.1 mm for lung and 6.1 ± 2.2 mm for heart. Subjective evaluation showed that the mesh-based approach could keep fine details, especially for the lung. The image-based approach seemed to overprocess contours and suffered from image resolution limits. CONCLUSION: We developed a practical approach for accurate contour deformation and demonstrated its effectiveness for both clinical and research applications.

Novel Hybrid Scattering- and Scanning-Beam Proton Therapy Approach.

PURPOSE: To determine whether a hybrid intensity-modulated proton therapy (IMPT) and passive scattered proton therapy (PSPT) technique, termed HimpsPT, could be adopted as an alternative delivery method for patients demanding scanning beam proton therapy. PATIENTS AND METHODS: We identified 3 representative clinical cases-an oropharyngeal cancer, skull base chordoma, and stage III non-small-cell lung cancer-that had been treated with IMPT at our center. We retrospectively redesigned these cases using HimpsPT. The PSPT plans for all three cases were designed with the same prescriptions as those used in the IMPT plans. In this way, the whole treatment was delivered using alternating or sequential PSPT and IMPT. RESULTS: All HimpsPT plans met the clinical dose criteria and were of similar quality as the IMPT plans. In the skull base case, the mixed plan was more effective at sparing the brain stem because the sharp penumbra of the aperture in the PSPT plans was not present in the IMPT plans. The HimpsPT plans were more robust than the clinical IMPT plans generated without robust optimization. CONCLUSION: The HimpsPT delivery technique can achieve a treatment-plan quality similar to that of IMPT, even in the most challenging clinical cases. In addition, at centers equipped with both scattering and scanning beam capabilities, the HimpsPT technique may allow more patients to benefit from scanning beam technology.