Two rare autosomal recessive neurological disorders identified by combined genetic approaches in a single consanguineous family with multiple offspring.

INTRODUCTION: Neurodevelopmental disorders (NDDs) refer to a broad range of diseases including developmental delay, intellectual disability, epilepsy, autism spectrum disorders, and attention-deficit/hyperactivity disorder caused by dysfunctions in tightly controlled brain development. The genetic backgrounds of NDDs are quite heterogeneous; to date, recessive or dominant variations in numerous genes have been implicated. Herein, we present a large consanguineous family from Turkiye, who has been suffering from NDDs with two distinct clinical presentations. METHODS AND RESULTS: Combined in-depth genetic approaches led us to identify a homozygous frameshift variant in NALCN related to NDD and expansion of dodecamer repeat in CSTB related to Unverricht-Lundborg disease (ULD). Additionally, we sought to functionally analyze the NALCN variant in terms of mRNA expression level and current alteration. We have both detected a decrease in the level of premature stop codon-bearing mRNA possibly through nonsense-mediated mRNA decay mechanism and also an increased current in patch-clamp recordings for the expressed truncated protein. CONCLUSION: In conclusion, increased consanguinity may lead to the revealing of distinct rare neurogenetic diseases in a single family. Exome sequencing is generally considered the first-tier diagnostic test in individuals with NDD. Yet we underline the fact that customized approaches other than exome sequencing may be used as in the case of ULD to aid diagnosis and better genetic counseling.

Functional characterization of KCNMA1 mutation associated with dyskinesia, seizure, developmental delay, and cerebellar atrophy.

KCNMA1 located on chromosome 10q22.3, encodes the pore-forming α subunit of the 'Big K+' (BK) large conductance calcium and voltage-activated K + channel. Numerous evidence suggests the functional BK channel alterations produced by different KCNMA1 alleles may associate with different symptoms, such as paroxysmal non kinesigenic dyskinesia with gain of function and ataxia with loss of function. Functional classifications revealed two major patterns, gain of function and loss of function effects on channel properties in different cell lines. In the literature, two mutations have been shown to confer gain of function properties to BK channels: D434G and N995S. In this study, we report the functional characterization of a variant which was previously reported the whole exome sequencing revealed bi-allelic nonsense variation of the cytoplasmic domain of calcium-activated potassium channel subunit alpha-1 protein. To detect functional consequences of the variation, we parallely conducted two independent approaches. One is immunostaining using and the other one is electrophysiological recording using patch-clamp on wild-type and R458X mutant cells to detect the differences between wild-type and the mutant cells. We detected the gain of function effect for the mutation (NM_001161352.1 (ENST00000286628.8):c.1372C > T;Arg458*) using two parallel approaches. According to the result we found, the reported mutation causes the loss of function in the cell. It should be noted that in future studies, it can be thought that the functions of genes associated with channelopathies may have a dual effect such as loss and gain.

Biallelic loss of TRAPPC9 function links vesicle trafficking pathway to autosomal recessive intellectual disability.

BACKGROUND: The trafficking protein particle (TRAPP) complex subunit 9 (C9) protein is a member of TRAPP-II complexes and regulates vesicle trafficking. Biallelic mutations in the TRAPPC9 gene are responsible for intellectual disability with expanded developmental delay, epilepsy, microcephaly, and brain atrophy. TRAPPC9-related disease list is still expanding, however, the functional effects of only a limited fraction of these have been studied. METHODS: In a patient with a pathological variant in TRAPPC9, clinical examination and cranial imaging findings were evaluated. Whole-exome sequencing, followed by Sanger sequencing was performed to detect and verify the variant. To confirm the functional effect of the mutation; variant mRNA and protein expression levels were evaluated by qRT-PCR and Western blotting. Immunostaining for TRAPPC9 and lipid droplet accumulation were examined. RESULTS: We have identified a novel homozygous c.696C>G (p.Phe232Leu) pathogenic variant in TRAPPC9 (NM_031466.6) gene as a cause of severe developmental delay. Functional characterization of the TRAPPC9 variant resulted in decreased mRNA and protein expression. The intracellular findings showed that TRAPPC9 protein build-up around the nucleus in mutant type while there was no specific accumulation in the control cell line. This disrupted protein pattern affected the amount of neutral lipid-carrying vesicles and their homogenous distribution at a decreasing level. CONCLUSION: Biallelic variants in the TRAPPC9 gene have been reported as the underlying cause of intellectual disability. This study provides functional evidence of the novel variant in TRAPPC9 We demonstrated that the loss of function variant exclusively targeting TRAPPC9 may explicate the neurological findings through vesicle trafficking.

Investigating differential miRNA expression profiling using serum and urine specimens for detecting potential biomarkers for early prostate cancer diagnosis

Background/aim: MicroRNAs (miRNAs) are known up-to-date candidate biomarkers for several diseases. In addition, obtaining miRNA from different body fluids such as serum, plasma, saliva, and urine is relatively easy to handle. Herein we aimed to detect miRNAs as biomarkers for early stage prostate cancer (PC). For this purpose, we used urine and serum samples to detect any significant differences in miRNA profiles between patients and healthy controls. Materials and methods: Total ribonucleic acid (RNA) in urine and serum samples were isolated from eight untreated PC patients, thirty healthy individuals were screened for miRNA profile, and candidate miRNAs were validated. Whole urinary and serum miRNA profile was analyzed using Affymetrix GeneChip miRNA 4.0 Arrays. Candidate miRNAs were investigated by stem-loop reverse transcription- polymerase chain reaction. Results: When we analyzed the urinary samples of PC patients, 49 miRNAs were detected to be upregulated and 14 miRNAs were found to be downregulated when compared with healthy controls. According to the serum samples, 19 miRNAs were found to be upregulated, and 21 miRNAs were found to be downregulated when compared with healthy individuals as well. Interestingly, we detected only four overlapping miRNAs (MIR320A, MIR4535, MIR4706, MIR6750) that commonly increase or decrease in both serum and urine samples. Among them, MIR320A was found to be downregulated, and MIR4535, MIR4706, and MIR6750 were found to be upregulated for urine samples. However, only MIR6750 was upregulated and the other three miRNAs were downregulated for serum samples. Conclusion: Notably, the expression profile of MIR320A was significantly altered in urine specimens of prostate cancer patients. We considered that MIR320A has been evaluated as a valuable biomarker that can be used in the early diagnosis of PC.

Expanding Clinical Phenotype of TRAPPC12-Related Childhood Encephalopathy: Two Cases and Review of Literature.

Biallelic mutations in the TRAPPC12 gene are responsible for early-onset progressive encephalopathy with brain atrophy and spasticity (PEBAS). To date, three different allelic variants have been reported. Next-generation sequencing allowed discovery of unique alternations in this gene with different phenotypes. We report two patients carrying TRAPPC12 variants, one previously reported and one unknown mutation, with severe neurodevelopmental delay and brain atrophy. Standard clinical examination and cranial imaging studies were performed in these two unrelated patients. In addition, whole-exome sequencing was performed, followed by Sanger sequencing for verification. The first patient, a 2-year-old boy, was found to be homozygous for the previously reported c.1880C > T (p.Ala627Val) mutation. He presented with a phenotype including severe progressive cortical atrophy, moderate cerebellar atrophy, epilepsy, and microcephaly, very similar to the previously reported cases. The second case, a 9-year-old boy, carried a novel homozygous c.679T > G (p.Phe227Val) variant and presented with mild cortical atrophy, severe cerebellar atrophy, and neither clinically manifest epilepsy nor microcephaly, which were previously considered typical findings in PEBAS with TRAPPC12 mutations. Our findings suggest that clinical and brain imaging findings might be more variable than previously anticipated; however, a larger number of observations would benefit for broader phenotypic spectrum.

Microencapsulated parathyroid allotransplantation in the omental tissue.

Permanent hypoparathyroidism is a severe clinical condition accompanied by low parathyroid hormone level. Conventional treatment requires lifelong medication, and daily drug usage has some side effects. To avoid this circumstance, transplantation is an alternative and curative option. Microencapsulation may be used as a transplantation approach particularly to evade immune response. In order to define treatment of permanent hypoparathyroidism, a 37-year-old female recipient who has permanent hypoparathyroidism was evaluated for 3 years. Routine tests, viral markers, and T and B lymphocyte cross-match tests were analyzed. In addition intradermal skin test was performed for ultrapure alginate. Microencapsulation of cultured parathyroid cells was performed with ultrapure alginate. Cell suspension was prepared and spheroids were generated with calcium chloride. Afterward, transplantation was performed with a laparoscopic approach in the omental tissue. The recipient was discharged from the hospital without complications. Serum calcium, parathyroid hormone (PTH), and phosphorus levels were observed throughout 1 year. During the follow-up period, no complications were observed. Serum calcium levels were increased significantly on day 10 and PTH levels were increased on day 25 as well. According to our knowledge, this is the first study where ultrapure alginate-based microencapsulated parathyroid cells were transplanted in the omental tissue. A significant increment of PTH levels was detected. Microencapsulated parathyroid cells showed the functionality of this technique for more than 1 year. This study showed that using ultrapure alginate-based microencapsulation without immunosuppression appears to be a promising technique.

Comparison of the efficacy of low-level laser therapy and photodynamic therapy on oral mucositis in rats.

Cancer treatment with chemotherapy or radiotherapy is associated with some side effects including in the oral cavity. One of the more significant oral complications is oral mucositis (OM) which induces severe pain and limits fundamental life behaviors such as eating, drinking, and talking. Although advancements in cancer treatment improved the survival rate, severe OM and opportunistic infection affect treatment adversely. Therefore, the control of OM is important for oral health quality of life and prognosis. Low-level laser therapy (LLLT) and photodynamic therapy (PT) are noninvasive methods that reduce inflammation and pain during wound healing. The aim of this study is to evaluate immunohistochemical and histological examination of the OM region of the PT comparing LLLT. In this study, 24 Sprague-Dawley rats were divided into three groups as control, LLLT, and PT groups. All groups received 5-fluorouracil intraperitoneally and a linear trauma to the mouth pouch with a needle. After the formation of OM in the mouth, the control group had no treatment; the LLLT group was administered LLLT, and the PT group had LLLT after indocyanine green application. Then all groups were sacrificed, and histological analyses and protein level detection of basic fibroblast growth factor (bFGF), transforming growth factor (TGF-ß), and platelet-derived growth factor (PDGF-BB) were evaluated in all groups. PT was determined to be more statistically significantly than LLLT with bFGF and PDGF-BB. However, regarding TGF-ß, no statistically significant difference was observed between the groups. Within the limitations of this study, indocyanine green may accelerate the LLLT effect. However, further studies on this subject are required.

Fresh tissue parathyroid allotransplantation with short-term immunosuppression: 1-year follow-up.

BACKGROUND: Permanent hypoparathyroidism is a serious problem and requires medications indefinitely. Parathyroid allotransplantation (PA) with short-term immunosuppression is definitive choice but long-term results are not clear. METHOD: We performed PA from two donors to two recipients. Both recipients were 39-year-old females. Donors were a 32-year-old female and a 36-year-old male, who both have chronic kidney disease. Routine tests, viral markers, and cross-matches were analyzed individually. The parathyroid glands were resected from the living donors, fragmented quickly in the operation room and injected into the left deltoid muscles of the two recipients. RESULTS: Methylprednisolone was administered on post-PA day one and two. Recipients were discharged from the hospital without complications. Calcium and PTH levels were observed throughout 1 year. We did not observe any complications during the follow-up period. Medications ceased in post-transplantation week 1 for Case 1 and after 1 month for Case 2. CONCLUSION: Fresh tissue PA with short-term immunosuppression appears to be a promising technique that is easy to perform, is cost-effective, has low risk of side effects and minimal complications with compatibility for HLA conditions. A longer follow-up period and more case studies are needed to determine the risks and benefits of this procedure for future cases.

Identification of suitable reference genes for RT-qPCR studies in human parathyroid tissue glandular cells.

Identifying a proper reference gene allows us to understand fundamental changes in many biological processes. Normalization during gene expression analyses is essential for every tissue/cell type, including parathyroid tissue glandular cells. Quantitative method of gene expression analyses via qRT-PCR method provides the accurate examination of every target gene. There are limited reports to present commonly used reference genes in human parathyroid tissues rather than for glandular cell types. This study aims to determine and compare the most stable to least stable genes for parathyroid tissue cells. 43 human parathyroid tissue obtained from primary and secondary hyperparathyroidism patients and glandular cells isolated enzymatically by the removal of extracellular matrix components. After extraction of the total RNA, cDNA synthesis was performed, then qRT-PCR evaluated 14 candidate reference genes. Stability was determined by RefFinder software (Delta ct, BestKeeper, Genorm, and NormFinder algorithms), and the outcome was evaluated for five groups. Even if assessed with different groups, the most stable genes were RPLP0 and GAPDH, while the CLTC and RNA 18S were the least stable. We have confirmed the comprehensive ranking of the most stable three genes alone with the NormFinder algorithm to understand intergroup variation and found out that RPLP0>GAPDH>PGK1. Lastly, comparisons of relative target gene (GCM2) expression revealed similar expression patterns for the most stable reference genes. The most stable reference gene is recommended for the stages where stability is evaluated using the results of four different approaches using RefFinder. We aspire for this study to assist future research to conduct thorough assessments of appropriate reference genes before engaging in gene expression analyses for parathyroid tissue.

An Alternative and Up-To-Date Recommendation for a Proper Site for Parathyroid Transplantation: The Omentum.

OBJECTIVES: Various sites have been used for parathyroid allotransplant, such as injection into the forearm, sternocleidomastoid, or deltoid muscles. However, transplant efficiency in these regions varies according to the results reported in the literature and also obtained from our previous studies. Using the omentum "as a natural incubator" for composite tissue-derived cell transplants to increase transplant success is possible. MATERIALS AND METHODS: To examine the efficiency of transplant sites for parathyroid allotransplant, we compared clinical cases from the literature and our experience with 23 cases. The omental transplant process is performed under general anesthesia by laparoscopic intervention. The abdominal cavity is visualized with an endocamera from a 5-mm trocar. With the help of a flexible catheter from another 5-mm trocar, microencapsulated or naked parathyroid cells are deposited on the omentum in approximately 30 mL of isotonic saline. The trocar sites are then sutured, and the surgery is terminated. Recipients were followed for 6 months with examination of biochemical parameters (parathormone, calcium, and phosphorus levels), medication use, and clinical scoring. RESULTS: In the 23 patients who received parathyroid allotransplants over the omentum, 7 recipients did not exhibit any changes according to the scoring system criteria. However, on average, serum calcium and phosphorus levels remained within the healthy reference range. In addition, use of oral medications among patients decreased, indicating successful transplant survival. These results were consistent with a significant decrease in hypocalcemia symptoms among patients. CONCLUSIONS: In the 23 patients, the success rate was 69.5% for the first 6-month period after transplant. Recent developments and large cohorts are needed to demonstrate the higher efficacy of parathyroid allotransplant over the omentum.

The Challenge of Preoperative Panel Reactive Antibody Positivity in Parathyroid Transplantation.

OBJECTIVES: Identifying suitable recipient criteria and matching recipients with appropriate donors are required to increase survival for parathyroid transplant. This study was undertaken to evaluate transplant survival rates while comparing preoperative panel reactive antibody positivity. MATERIALS AND METHODS: The study included 14 hypoparathyroidism patients who presented to our clinic for parathyroid transplant. Preoperative ABO compatibility and negative cross-match tests were prioritized for recipient-donor matching, and panel reactive antibody screening tests were performed. During the 24-month follow-up, we evaluated medication use and serum calcium, phosphorus, and parathormone levels of patients. RESULTS: Preoperative panel reactive antibody positivity was assessed in 3 groups. The HLA class I-positive group (mean fluorescence intensity range, 179-1770) showed decreased medication use and stability in serum calcium levels. The HLA class IIpositive (mean fluorescence intensity range, 85-3959) showed decreased medication use by 25% to 50% and returned to their former prescription doses after 12 months. An opposite pattern was observed in 2 patients with panel reactive antibody positivity for both HLA classes (mean fluorescence intensity range, 462-2289), with 1 patient requiring medication for continuing symptoms and the other patient occasionally taking additional magnesium supplementation, despite decreased medication doses after 12 months. Serum calcium levels remained normal, and parathormone and phosphorus levels were elevated. CONCLUSIONS: Improving patient symptoms and having no requirement for intravenous calcium replacement are priorities, and monitoring serum levels is the next important step. Varied panel reactive antibody positivities and survival rates indicate a requirement, and each HLA class could require a proper limitation for the mean fluorescence intensity. Preoperative mean fluorescence intensity cut-off value should be <900. Higher mean fluorescence intensity values in panel reactive antibody screenings could increase risk of short-term graft survival after parathyroid transplant. Further studies should include immunological risk assessments by individualizing the outcome with donor-specific antibodies.

GLUT-Targeting Phototherapeutic Nanoparticles for Synergistic Triple Combination Cancer Therapy.

The combination of multimodal therapies into one nanocarrier system is promising for its potential to enhance treatment performance by overcoming the efficacy problems encountered in conventional monomodal therapy. In this study, targeted and multimodal therapeutic hybrid nanocarriers are fabricated for breast cancer treatments. In this context, the synthesized gold nanorods (AuNRd), photothermal therapy (PTT) agent, are coated with doxorubicin (DOX) conjugated, targeted, and biocompatible tetrablock glycopeptide (P(DMAEMA-b-HMBAMA-b-FrucMA)-b-P(Lys)/DOX, P-DOX) polymer. Here, fructose-based (Fruc) glycopeptide polymer enhances cellular uptake into breast cancer through GLUT5. A photosensitizer molecule, indocyanine green (ICG), was loaded into the particles to provide photodynamic therapy (PDT) upon NIR light at 808 nm. In the final step of the fabrication, the polymer-coated nanoparticles are integrated with antisense ISIS5132 oligonucleotides to prevent apoptotic resistance of cells against drug molecules. The biocompatibility and therapeutic efficacy of the nanoparticles are evaluated on both human normal skin fibroblast cell (CCD-1079Sk) and human breast cancer cell (MCF7) lines. These multimodal therapeutic AuNRd@P-DOX/ICG/ISIS5132 nanoparticles demonstrate an efficient triple synergistic effect of chemo-/PTT/PDT, which is desired for breast cancer treatment. We believe that this promising multimodal therapeutic nanoparticle system can promote the further clinical application in the treatment of breast cancer and can also be adapted to other types of cancer.

HLA Class I Expression Changes in Different Types of Cultured Parathyroid Cells.

OBJECTIVES: Tissue-specific immunogenicity can be characterized by the determination of human leukocyte antigens (HLA). Parathyroid hyperplasia tissue cells are presumed to have the ability to lose HLA class I expression profile during cultivation, whereas healthy parathyroid cells are presumed to already express HLA class I molecules at low levels. However, there are conflicting results about the expression of HLA class I antigens. In this study, our aim was to evaluate different patterns of HLA class I expression in different parathyroid tissue cells. MATERIALS AND METHODS: Parathyroid tissue cells were isolated enzymatically and cultured in vitro. Expression of HLA class I (HLA-A, HLA-B, HLA-C) mRNA and protein levels were studied in 7 parathyroid adenomas and 9 parathyroid hyperplasia tissue samples by reverse transcriptase-polymerase chain reaction and Western blot analyses. RESULTS: HLA-A protein expression remained stable in parathyroid adenoma and hyperplasia tissue, but HLA-A mRNA expression decreased in adenoma tissue. In parathyroid hyperplasia tissue, HLA-B protein expression remained stable, although mRNA expres-sion levels decreased during cultivation. HLA-C mRNA expression was steady in parathyroid adenoma yet significantly decreased in hyperplasia tissue samples. HLA-C protein expression levels were below 30 pg for both types of parathyroid tissue during cultivation. CONCLUSIONS: HLA class I expression levels of para-thyroid hyperplasia and adenoma tissue were not found to be similar. Parathyroid hyperplasia tissue is the donor tissue for the treatment of permanent hypoparathyroidism. Therefore, expression patterns of HLA class I are directly relevant to the transplant process. In particular, the HLA region is highly polymorphic, and, as a consequence of this, heterogeneous correlations among HLA-A, HLA-B, and HLA-C expression patterns of parathyroid tissue should be evaluated in detail before transplant for future studies.

Sulforaphane, a Chemopreventive Compound Induces Necrotic Behavior and Inhibits S-phase of Cell Cycle in Human Kidney Cells in Vitro.

Sulforaphane (SFN) is an organosulfur product of found isothiocyanates in vegetables. The chemopreventive effects of SFN have revealed that there is a link between excessive consumption of SFN-rich vegetables and cancer formation without possible toxicological consequences. We aimed to evaluate the cellular outcome of SFN from a toxicological perspective, particularly for renal cells including clear cell adenocarcinoma (769-P) and human embryonic renal epithelial (293T) cells. The viability/cytotoxicity experiments were performed with methyl thiazole diphenyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays. IC50-dependent, non-cytotoxic concentrations were used for the determination of cell cycle status and apoptosis by using flow cytometry and western blot. A certain concentration of SFN effectively altered apoptotic/necrotic behavior in 769-P compared to the control group 293T. Cell cycle status remained stable while showing a decreased proliferation profile for 769-P cells. The percentage of the S phase from the cell cycle in 293T cells significantly reduced without affecting proliferation status. The use of SFN as an alternative to traditional treatments might be considered for the battle against renal cell carcinoma but the current findings showed that caution should be applied particularly for renal cells. Our study will provide a basis for future in vivo studies to support traditional cancer therapies.

HLA-DR, -DP, -DQ expression status of parathyroid tissue as a potential parathyroid donor selection criteria and review of literature.

BACKGROUND: Basic and clinical studies about parathyroid allotransplantation have to be utilized with more definitive criteria for longer graft survival. Several reports demonstrated different isolation and cultivation methods for parathyroid cells to minimize their immunogenicity. In this study, we aim to compare and evaluate the clinical characteristics and the status of HLA class II expression changes in parathyroid tissue. METHODS: A total of 22 parathyroid hyperplasia tissue donors was included in this study. Clinical characteristics were evaluated and compared with the HLA-DR, -DP, -DQ mRNA, and protein expression levels which were determined by qRT-PCR and Western blot. RESULTS: We have compared the clinical characteristics (age, dialysis duration, frequency, recurrency of hyperparathyroidism and, calcimimetic usage) and HLA class II expression. HLA class II mRNA and protein levels showed varied expression patterns between tissues. Only, the HLA-DP has high mRNA expression levels without affecting the protein level when compared with the ages of the tissue donors. In addition, the HLA-DR, HLA-DP, and HLA-DQα1 protein expression levels showed a permanent and varied expression rate between tissues. CONCLUSION: Expression of HLA class II molecules in parathyroid cells appears to constitute a decisive factor. Despite the lack of clinical outcomes, present data proposes new insight with a detailed understanding of parathyroid immunogenicity. In the future, randomized controlled clinical trials are needed for the accurate assessment of the effect of the varied HLA class II expression profiles in parathyroid tissue.

Graft survival effect of HLA-A allele matching parathyroid allotransplantation.

Permanent hypoparathyroidism is an endocrine disease that is mostly associated with the disruption of the parathyroid glands during surgery. Allotransplantation is the most promising approach for treatment particularly for its cost-effective and exact curative potential. Herein our aim was to evaluate human leukocyte antigen (HLA)-A allele matching effect on clinical improvement and graft survival after parathyroid transplantation. We performed parathyroid transplantation between ABO/Rh compatible recipient and an unrelated donor who has chronic kidney disease. Preoperative immunological tests include panel reactive antibody, T-flow cytometry crossmatch, B-flow cytometry crossmatch, autoflow cytometry crossmatch, and complement-dependent cytotoxicity crossmatch tests were performed. After histopathological evaluation, half of the resected parathyroid gland cells were isolated and transplanted to the omentum surface by laparoscopy. The transplantation outcome was followed up throughout 382 days. The recipient discharged 2 days after transplantation without any complication. During follow-up, calcium and vitamin D supplementation reduced to a one-third dose; even the intact PTH levels remained low. However, clinical improvement was observed by serum calcium levels. The recipient still continues with low-dose supplementation after 382 days of post-transplantation. Parathyroid cell transplantation to the omental tissue is the most promising option even with only one allele matching for patients with using lifelong high-dose supplementation. Clinical improvements and long-term effect of HLA-A allele matching should be evaluated with more studies and in larger cohorts as well.

Xenotransplantation of Microencapsulated Parathyroid Cells as a Potential Treatment for Autoimmune-Related Hypoparathyroidism.

OBJECTIVES: Hypoparathyroidism occurs as a part of a complex autoimmune syndrome or iatrogenically after neck surgery. The disease presents many challenges, such as hypocalcemia, hyperphosphatemia, and low/undetectable parathormone levels. Allotrans - plantation of parathyroid tissue or cells has been reported as a promising option to overcome these effects. Transplantation of microencapsulated parathy - roid tissue or cells offers an immune escape, which particularly restores the parathyroid function for autoimmune-related hypoparathyroidism. So far, clinical and in vivo studies have demonstrated limited graft survival and instability for the available biocompatible materials. In addition, the transplant site, proper local isolation, and biocompatibility of materials are directly related to survival rate. MATERIALS AND METHODS: A microencapsulated parathy - roid xenotransplant model by using high guluronic acid-containing ultrapure alginate transplanted into rat omentum was tested in vivo for 1 year. RESULTS: After stability of empty microcapsules was ensured, parathyroid cells were microencapsulated and transplanted in rats, with results compared versus rats with naked (nonencapsulated) parathyroid cells (both groups followed for 64 weeks). Rats remained normocalcemic, and preinflammatory cytokine levels showed dramatic changes. Despite a delay posttransplant, parathormone levels increased significantly. All retrieved microencapsules elicited pericapsular fibrotic overgrowth; however, the fibrosis area was shown to be well tolerated. CONCLUSIONS: The possible role of accumulation/cell infiltration of immune response remains to be elucidated. In conjunction with this, the use of nonencapsulated parathyroid cells was also positively correlated with survival rates. A similar evaluation using ultrapure alginate materials and omental transplantation may enable the future determination for the long-term effects of correction of parathor - mone insufficiency in patients with severe hypocalcemic responses and other endocrine diseases.

Differential anti-proliferative and apoptotic effects of lichen species on human prostate carcinoma cells.

Lichens are stable symbiotic associations between fungus and algae and/or cyanobacteria that have different biological activities. Around 60% of anti-cancer drugs are derived from natural resources including plants, fungi, sea creatures, and lichens. This project aims to identify the apoptotic effects and proliferative properties of extracts of Bryoria capillaris (Ach.) Brodo & D.Hawksw, Cladonia fimbriata (L.) Fr., Evernia divaricata (L.) Ach., Hypogymnia tubulosa (Schaer.) Hav., Lobaria pulmonaria (L.) Hoffm., and Usnea florida (L.) Weber ex Wigg. lichen species on prostate cancer cells. Lichen extracts were performed by ethanol, methanol, and acetone separately by using the Soxhlet apparatus and the effects of the extracts on cell viability, proliferation, and apoptosis were measured with the utilization of MTT, LDH assay, Annexin V assay, and Western Blot. Findings of our study revealed a positive correlation between the elevation of cell sensitivity and the increase in the treatment doses of the extract in that higher doses applied reverberate to higher cell sensitivity. A similar correlation was also identified between cell sensitivity elevation and the duration of the treatment. Evidence in our study have shown the existence of an anti-proliferative effect in the extracts of Bryoria capillaris, Evernia divaricata (L.) Ach., Hypogymnia tubulosa (Schaer.) Hav., Lobaria pulmonaria (L.) Hoffm., and Usnea florida (L.) Weber ex Wigg., while a similar effect was not observed in the extracts of Cladonia fimbriata. Evernia divaricata induced anti-proliferative and apoptotic effects in PC-3 cells, which induced apoptotic cell death by both extrinsic and intrinsic pathways. Hypogymnia tubulosa has been shown to have anti-proliferative and apoptotic effects in all extractions methods and our findings identified that both the percentage of the apoptotic cells and apoptotic protein expressions recorded an increase at lower treatment concentrations. Although Lobaria pulmonaria is known to have significant cytotoxic effects, we did not observe a decrease in cell proliferation. Indeed, proliferation marker proliferating cell nuclear antigen (PCNA) protein expression levels have shown an increase in all extracts, while Usnea florida exhibited apoptosis induction and slight proliferation reduction in extract treatments with lower concentrations. We tested 18 extracts of six lichen species during our study. Of these, Evernia divaricata and Hypogymnia tubulosa demonstrated significant apoptotic activity on prostate cancer cells including at low concentrations, which implies that it is worth pursuing the biologically active lead compounds of these extracts on prostate cancer in vitro. Further corroboratory studies are needed to validate the relative potential of these extracts as anti-metastatic and anti-tumorigenic agents.

Correction: Differential anti-proliferative and apoptotic effects of lichen species on human prostate carcinoma cells.

[This corrects the article DOI: 10.1371/journal.pone.0238303.].

Discharging a Patient Treated With Parathyroid Allotransplantation After Having Been Hospitalized for 3.5 Years With Permanent Hypoparathyroidism: A Case Report.

INTRODUCTION: Parathyroid allotransplantation is one of the methods used in the treatment of permanent hypoparathyroidism. We present a patient who underwent continuous intravenous (IV) calcium replacement therapy because of permanent hypoparathyroidism after total thyroidectomy. CASE PRESENTATION: A 47-year-old woman who underwent a total thyroidectomy with a multinodular goiter developed hypoparathyroidism and hypocalcemia 1 week after discharge. The patient was started on daily oral calcitriol, magnesium effervescent, vitamin D, and IV calcium gluconate and was unable to be discharged because the IV calcium could not be stopped. After 3.5 years, 50×106 parathyroid cells were transplanted by injecting the cells into the left deltoid muscle of the patient. The immunosuppression of the patient, who used 20-mg methylprednisolone for the first month, was completely discontinued. RESULTS: No complications were observed in the patient after transplantation. The parenteral calcium replacement of the patient was progressively interrupted after transplantation. The patient's serum calcium level was 7.8 mg/dL and the PTH level was 6.9 pg/mL without IV calcium replacement at 12 weeks after transplantation, and the patient was discharged using oral ionized calcium. In the 10-week follow-up after discharge, the need for IV calcium was not observed in the patient. CONCLUSION: Parathyroid allotransplantation is a simple, fast, and cost-effective method that should be tried in patients with persistent hypoparathyroidism, especially those who have to take IV calcium replacement, because its cost is much cheaper than standard medical care, its morbidity is much more limited, and it increases the patient's quality of life.