Conceptos de criobiología y fisiología ovárica en la preservación de la fertilidad / Concepts of cryobiology and ovarian physiology in fertility preservation

La criobiología se enfoca en entender cómo reaccionan los materiales biológicos a temperaturas muy bajas. Este campo ha experimentado avances significativos, particularmente en el ámbito de la reproducción asistida, donde se han desarrollado programas para preservar la fertilidad. Estos desarrollos revisten importancia crítica para quienes exploran alternativas en materia de fertilidad y preservación de gametos. Por otro lado, la preservación de la fertilidad tiene como objetivo proteger la capacidad reproductiva de una persona por diferentes condiciones de salud, tratamientos médicos o razones sociales que la puedan comprometer. Las técnicas aceptadas para la preservación de fertilidad en humanos son la criopreservación de gametos y de embriones. Existe evidencia prometedora creciente sobre distintas técnicas experimentales dentro de este campo, como la crioconservación del tejido gonadal, o estrategias de maduración in vitro, así como nuevas metodologías en los protocolos criogénicos que supondrán una optimización de los resultados y un punto de inflexión dentro del campo de la reproducción asistida. Este trabajo tiene como objetivo explorar el estado del arte de las estrategias actuales ofrecidas a las mujeres en el contexto de preservación de la fertilidad, revisar los avances en criobiología y su papel en la evolución de este ámbito.(AU)

Cryobiology focuses on understanding how biological materials react to very low temperatures. This field has experienced significant advances, particularly in the field of assisted reproduction, where programs have been developed to preserve fertility. These developments are of critical importance for those exploring alternatives in fertility and gamete preservation. Fertility preservation aims to protect a person's reproductive capacity under various health conditions, medical treatments, and social reasons that may compromise it. Accepted techniques for human fertility preservation include the cryopreservation of gametes and embryos. There is growing promising evidence on different experimental techniques within this field, such as cryopreservation of gonadal tissue or in vitro maturation strategies, as well as new methodologies in cryogenic protocols that will optimize results and mark a turning point in the field of assisted reproduction. This work aims to explore the current state-of-the-art strategies offered to women in the context of fertility preservation, review advances in cryobiology, and its role in the evolution of this area.(AU)

Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research.

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2 SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7 M Me2 SO and 100 mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics.

Cryobiology and fertility preservation: a perspective on past, current and future studies.

Cryopreservation has been used over many decades for the maintenance of viable biological specimens. Its expansion into the area of fertility preservation has been a natural outcome of the increased risks to human fertility from diseases, such as cancer and its treatment protocols, including radiation and chemo-therapy, and the general lifestyle trend to later marriages. The use of assisted reproductive techniques (ART) in preserving fertility have benefitted significantly from new scientific approaches, such as cryostorage, in which live cells and tissues are stored at low temperatures and revived when necessary. This review focuses on "cryopreservation science monitoring in reproductive biomedicine" to evaluate knowledge, trends, driving forces, impetus, and emerging technologies in order to draw a future roadmap for this field. Our analysis of the field of cryobiology emphasizes the significance of strategic planning of cryobiology research to support more its extensive use in therapeutics in the future. The Royan Institute (Tehran, Iran) recognises this need and has developed a strategic plan to engage in multidisciplinary research on the application of cryobiology, including cryobioengineering, in disease mitigation. We hoped that this study can help improve the quality and quantity of public discourse and expert awareness of the role for cryopreservation in fertility preservation within ART. DOI: 10.54680/fr23410110112.

Implementation of transbronchial lung cryobiopsy in a tertiary referral center for interstitial lung diseases: a cohort study on diagnostic yield, complications, and learning curves.

BACKGROUND: Transbronchial lung cryobiopsy (TBLC) has been introduced as an alternative to surgical lung biopsy (SLB) in the diagnostics of interstitial lung diseases (ILD). Despite controversy on safety, TBLC is increasingly implemented in ILD centers with an apparent diagnostic yield comparable to SLB. The aim of this study was to assess TBLC implementation experiences from a tertiary Danish ILD center regarding diagnosis, complications, and learning curves for TBLC performance. METHODS: TBLC was prospectively performed in a cohort of patients with unclassifiable ILD based on a preceding multidisciplinary clinical and radiological revision. TBLC was performed as an outpatient procedure with the patients in general anesthesia using a flexible bronchoscope with 1.9 or 2.4 mm cryoprobes. Learning curves for TBLC performance were calculated using cumulated sum (CUSUM) scores for diagnostic yield, pneumothorax, and bleeding. RESULTS: From February 2017 to March 2020 141 patients (86 (61%) men, median age 69 years [IQR, 60-74 years]) had TBLC performed. A histological and confirmative diagnosis was made in 101 patients (75.2%) and 124 patients (87.9%, i.e. clinical diagnostic yield), respectively, in whom idiopathic interstitial pneumonias constituted the majority (67.3%) of the clinical diagnoses. We observed 2 deaths (1.4%) within 30 days of TBLC, but no procedure-related mortality or severe bleeding. Moderate bleeding occurred in 23 patients (16.3%), pneumothorax in 21 patients (14.9%) with only 14 patients (9.9%) requiring a pleural drain. Based on the CUSUM score analysis, the diagnostic yield obtained was satisfactory throughout the period. CONCLUSION: This study reports experiences of outpatient TBLC implementation in a tertiary referral ILD center from the largest investigated TBLC cohort in Scandinavia The diagnostic yield and prevalence of complications obtained by TBLC from this single center study on unclassifiable ILD support outpatient TBLC as a valuable and safe alternative to SLB to diagnose ILD in well-selected patients. The learning curves for TBLC were acceptable in the hands of experienced bronchoscopists.

Hypothermic storage of leukoreduced red blood cells for greater than 21 days is a safe alternative to irradiation.

BACKGROUND: Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS: Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS: Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS: Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.

Reconstrucción de precipitación y temperatura con anillos de crecimiento anual del ciprés Taxodium mucronatum (Taxodiaceae) en Coahuila, México / Reconstruction of precipitation and temperature with annual growth rings of the cypress Taxodium mucronatum (Taxodiaceae) in Coahuila, Mexico

Introducción: La determinación del clima en cuencas productoras de agua, como la del río Sabinas, Coahuila, donde no existe información de su variabilidad, se puede estimar mediante anillos de crecimiento de ahuehuete (Taxodium mucronatum Ten.), especie longeva con anillos bien definidos, presente en las zonas ribereñas, constituye un "proxy" o método indirecto de la variabilidad climática interanual y multianual. Objetivo: Desarrollar una reconstrucción estacional de precipitación y temperatura máxima y analizar la influencia que ejercen fenómenos de circulación global en el crecimiento de la especie. Métodos: Los núcleos de crecimiento de T. mucronatum fueron datados a través de técnicas dendrocronológicas para producir una serie de ancho de anillo. Se utilizaron datos de mallas del clima de dos bases de datos para desarrollar el análisis de la función de respuesta con fines de reconstrucción climática. Resultados: Una cronología de anillo total de 218 años (1808-2018) se desarrolló con especímenes de T. mucronatum en parajes del Río Sabinas, con la que se generó una reconstrucción estacional de precipitación abril-junio y de temperatura máxima junio-julio. El periodo detectado más seco se presentó de 1815 a 1818 con una precipitación de 27.39 mm y una temperatura máxima de 41.16 °C; mientras que el año más húmedo fue 1828 con 393.72 mm. La cronología de anillo total se correlacionó con los índices de sequía SPEI y PDSI, y con índices de fenómenos atmosféricos como El Niño Oscilación del Sur, a través del Índice de Oscilación del Sur (SOI) y el índice Multivariado (MEI); Oscilación Decadal del Pacífico (PDO) y Oscilación Multidecadal del Atlántico (AMO). La relación entre el índice de anillo total y el del Índice Estandarizado de Precipitación Evaporación (SPEI) mostró significancia en el mes de junio (r = 0.52, P < 0.01), al igual que el Índice de Severidad de Sequía del Palmer (PDSI), PDSI anual (r = 0.38, P < 0.05). El SOI reconstruido noviembre-febrero, se asoció significativamente con la serie dendrocronológica (r = -0.41, P < 0.01). La Oscilación Decadal del Pacífico y la Oscilación Multidecadal del Atlántico, no mostraron significancia. Conclusiones: En este estudio, desarrollamos una función de respuesta climática y reconstruimos variables climáticas estacionales (precipitación, temperatura máxima) de importancia para desarrollar estrategias de manejo para la conservación de T. mucronatum en esta cuenca, e implementar acciones de mitigación para la presencia de eventos climáticos extremos que se pueden presentar en los próximos años.

Introduction: Dendroclimatic reconstructions in water-yield basins lacking hydroclimatic data, such as the Rio Sabinas is important to analyze its interannual and multiannual climatic variability. One of the species useful for this purpose is the Montezuma baldcypress (Taxodium mucronatum Ten.), a long-lived species with well-defined annual rings, present along the riparian zone of the Rio Sabinas that constitutes a "proxy" of interannual and multiannual climate variability. Objective: Develop a seasonal precipitation and maximum temperature reconstructions, and to analyze the influence of global circulatory modes on the species annual radial increase. Methods: Increment cores of the Montezuma baldcypress specimens were dated through dendrochronological techniques to produce a ring-width series. Climate gridded data from two databases were used to develop a response function analysis for climate reconstruction purposes. Results: A ring-width chronology extending from 1808 to 2018 (211 years) was developed and used to develop a seasonal April-June precipitation and a mean June-July maximum temperature reconstruction. The driest period detected on the rainfall reconstruction occurred from 1815 to 1818 with 27.4 mm and a maximum temperature of 41.2 °C; while the wettest year was 1828 with 393.72 mm. The ring-width chronology was correlated with the Standardized Precipitation Evaporation Index (SPEI) and the Palmer Drought Severity Index (PDSI), and with indices of atmospheric phenomena such as El Niño Southern Oscillation (ENSO), through the Southern Oscillation Index (SOI) and the Multivariate Index (MEI); Pacific Decadal Oscillation (PDO), and the Atlantic Multidecadal Oscillation (AMO). The relationship between the ring-width series and drought indices (SPEI, PDSI) was significant in June (r = 0.52, P < 0.01), and June-August (r = 0.38, P < 0.05) for the SPEI and reconstructed PDSI, respectively. It was found a significant association between the ring-width chronology and the reconstructed November-February SOI (r = -0.41, P < 0.01). The Pacific Decadal Oscillation and the Atlantic Multidecadal Oscillation were not significant. Conclusions: On this study, we developed a climatic response function and reconstructed seasonal climatic variables (precipitation, maximum temperature) of importance to develop management strategies for conservation of the Montezuma bald cypress on this basin, and to implement mitigation actions for the presence of extreme climatic events that may occur in coming years.

Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity?

Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice for most animal cell systems since the early history of cryopreservation. It has been used for decades in many thousands of cell transplants. These treatments would not have taken place without suitable sources of DMSO that enabled stable and safe storage of bone marrow and blood cells until needed for transfusion. Nevertheless, its effects on cell biology and apparent toxicity in patients have been an ongoing topic of debate, driving the search for less cytotoxic cryoprotectants. This review seeks to place the toxicity of DMSO in context of its effectiveness. It will also consider means of reducing its toxic effects, the alternatives to its use and their readiness for active use in clinical settings.

¿Es necesaria la realización de ecocardiograma en la valoración de hipertensión pulmonar previa a una criobiopsia transbronquial? / Transbronchial cryobiopsy, is essential to rule out pulmonary hypertension?

INTRODUCCIÓN: El objetivo de nuestro estudio fue valorar la posibilidad de diagnosticar la Hipertensión Pulmonar (HP) mediante signos en la tomografía computarizada (TC), en pacientes con EPID que van a ser sometidos a criobiopsia transbronquial (CBxTb) sin necesidad de realizar ecocardiograma transtoracico (EcoC) sistemático y si la existencia de HP en TC influye de manera relevante en el riesgo de complicaciones de sangrado y en las demoras de realización de la técnica. MATERIAL Y MÉTODOS: Estudio prospectivo de casos controles de todos los pacientes en los que se realizó CBxTb en un período de 20 meses. Los controles fueron los pacientes en los que se realizó CBxTb con EcoC previa protocolizada y los casos los pacientes en los que se realizó EcoC pre CBxTb sólo sin había signos de HP en la TC. Analizamos la correlación entre los datos de HP de la TC y EcoC, las complicaciones por sangrado y la demora al procedimiento entre los grupos. RESULTADOS: Se han realizado 40 CBxTb. Se incluyeron 16 controles; 12 (75%) sin signos de HP en la TC con una especificidad de ésta en relación al EcoC del 85%, sensibilidad 71% y VPN 82%. Se incluyeron 24 casos y, tras los hallazgos de la TC, sólo se solicitó EcoC preCBxTb a 4 pacientes, de los que 3 resultaron falsos positivos. No hubo diferencias entre grupo de controles y casos en cuanto al sangrado (18,7% vs. 16,7%; p= 0,54) pero si en la demora diagnóstica (62 vs. 37; p= 0,03). CONCLUSIÓN: En nuestra serie de pacientes con EPID y CBxTb consideramos indicada la realización de EcoC sólo en casos con signos de HP en la TC, permitiendo acortar la demora diagnóstica sin aumento de sangrados

INTRODUCTION: Our objective was to assess whether the performance of EcoC only in those patients in which the caliper measurement of the PA on CT suggested the existence ofPAH, relevant influence on the risk of bleeding complications and delays CbxTb conducting. MATERIAL AND METHODS: It has been conducted prospective, case-control, of all patients who underwent CbxTb in our unit over a period of 20 months. Defining control group those patients who EcoC was done protocolised, before CbxTb, and cases all patients who EcoC was made only in cases where there were signs of PAH in CT. We analized the correlation in HP diagnosis signs between CT and EcoC data, risk of bleeding and delays in the procedure. RESULTS: We were performed 40 patients. In the control group include 16 patients, CTdetected no signs of HP in 12 (75%) cases, with a specificity of 85%, sensitivity of 71% and negative predictive value of 82%. In our cases group, we include 24 patients, and EcoC was only made before the procedure in 4 cases, 3 of them false positives. No differences in bleeding was found between control and cases groups (18.7% vs. 16.7%; p= 0.54) but for the delays in the procedure was lower in the cases group (62 days vs. 37; p= 0.03) .Conclusion. In our study with interstitial lung disease and CbxTb we conclude that EcoC was indicated only in the cases with PH findings on CT reducing the delays in the procedure without bleeding rate increase

The International Teaching and Practice of Cryobiology and Biobankology Course in China.

In the past 10 years, clinical biobanks have experienced increasing expansion in China. Demand for systematically educated biobanking professionals is a priority for Chinese biobanks' agenda. The cryobiology and biobankology course is the first semester-long course in China, designed and developed at Central South University with international cooperation. Leading professors were from China, the United States, United Kingdom, and Canada to teach the latest version of biobanking knowledge and skills around the globe. This course is a comprehensive elective course with six specific teaching modules, which is suitable for graduate students majoring in basic medical sciences, clinical medicine, life sciences, mechanical engineering, and biomedical engineering, who would like to seek biobanking careers in the future. Participants from China, Czech Republic, Ghana, Madagascar, Tanzania, South Sudan, and Israel attended the course. Through taking this course, students can broaden their international academic horizons and cultivate the ability to learn and apply the knowledge of biology, medicine, and engineering to analyze and explain the low-temperature biology and clinical samples-based research practice. At the same time, the course enables students to realize the importance of multidisciplinary fields of biobanking and the significance of innovative precision medicine research, and further enlightens students' enthusiasm to pursue biobanking professional careers, and in the future they can proudly call themselves "biobankers."

Cryopreservation and transplantation of amputated finger.

Every year tens of millions reconstruction surgeries were performed to repair tissue defects caused by trauma or malignant tumor. As the composite tissues are very difficult to be preserved for a long time in vitro, "organ bank" was proposed. In addition to immune rejection, tissue cryopreservation is the key step of the "Organ Bank". In this study, two severed fingers were cryopreserved because the patients' conditions were not allowed to undergo the replantation immediately. The fingers were perfused with the cryopreservation solution through the digital artery at a speed of 1 ml/min, they were frozen following a control rate freezing procedure and stored in the liquid nitrogen. One finger was cryopreserved for 10 days and the other for 30 days. Both fingers were successfully replanted when the patient get better. After 1 year follow-up, the replanted fingers achieved satisfactory appearance and function. Our result demonstrate small composite tissues such as severed fingers can be cryopreserved and replanted successfully, which will help the patients who cannot receive the replantation immediately. This method will have important application value in cryobiology and clinical medicine.

Low Temperature Promotes Anthocyanin Biosynthesis and Related Gene Expression in the Seedlings of Purple Head Chinese Cabbage (Brassica rapa L.).

To elucidate the effect of low temperature on anthocyanin biosynthesis in purple head Chinese cabbage, we analyzed anthocyanin accumulation and related gene expression in the seedlings of purple head Chinese cabbage, white head parent Chinese cabbage, and its purple male parent under a normal 25 °C temperature and a low 12 °C temperature. Anthocyanin accumulation in purple lines was strongly induced by low temperature, and the total anthocyanin content of seedlings was significantly enhanced. In addition, nearly all phenylpropanoid metabolic pathway genes (PMPGs) were down-regulated, some early biosynthesis genes (EBGs) were up-regulated, and nearly all late biosynthesis genes (LBGs) directly involved in anthocyanin biosynthesis showed higher expression levels in purple lines after low-temperature induction. Interestingly, a R2R3-MYB transcription factor (TF) gene 'BrMYB2' and a basic-helix-loop-helix (bHLH) regulatory gene 'BrTT8' were highly up-regulated in purple lines after low temperature induction, and two negative regulatory genes 'BrMYBL2.1' and 'BrLBD38.2' were up-regulated in the white line. BrMYB2 and BrTT8 may play important roles in co-activating the anthocyanin structural genes in purple head Chinese cabbage after low-temperature induction, whereas down-regulation of BrMYB2 and up-regulation of some negative regulators might be responsible for white head phenotype formation. Data presented here provide new understanding into the anthocyanin biosynthesis mechanism during low temperature exposure in Brassica crops.

A comparison of low temperature biology of Pieris rapae from Ontario, Canada, and Yakutia, Far Eastern Russia.

Low temperatures limit the distribution and abundance of ectotherms. However, many insects can survive low temperatures by employing one of two cold tolerance strategies: freeze avoidance or freeze tolerance. Very few species can employ both strategies, but those that do provide a rare opportunity to study the mechanisms that differentiate freeze tolerance and freeze avoidance. We showed that overwintering pupae of the cabbage white butterfly Pieris rapae can be freeze tolerant or freeze avoidant. Pupae from a population of P. rapae in northeastern Russia (Yakutsk) froze at c. -9.3 °C and were freeze-tolerant in 2002-2003 when overwintered outside. However, P. rapae from both Yakutsk and southern Canada (London) acclimated to milder laboratory conditions in 2014 and 2017 froze at lower temperatures (< -20 °C) and were freeze-avoidant. Summer-collected P. rapae larvae (collected in Yakutsk in 2016) were partially freeze-tolerant, and decreased the temperature at which they froze in response to starvation at mild low temperatures (4 °C) and repeated partial freezing events. By comparing similarly-acclimated P. rapae pupae from both populations, we identified molecules that may facilitate low temperature tolerance, including the hemolymph ice-binding molecules and several potential low molecular weight cryoprotectants. Pieris rapae from Yakutsk exhibited high physiological plasticity, accumulating cryoprotectants and almost doubling their hemolymph osmolality when supercooled to -15 °C for two weeks, while the London P. rapae population exhibited minimal plasticity. We hypothesize that physiological plasticity is an important adaptation to extreme low temperatures (i.e. in Yakutsk) and may facilitate the transition between freeze avoidance and freeze tolerance.

Foundations of modeling in cryobiology-III: Inward solidification of a ternary solution towards a permeable spherical cell in the dilute limit.

In the previous two manuscripts we outlined the general theory of heat and mass transport in a cell-liquid-ice system with general boundaries and nonideal and nondilute assumptions. Here we simplify the models considerably by presenting a reduction to a spherically symmetric system-a spherical cell with an encroaching spherical ice front. We also reduce to linear approximations of the nonideal nondilute models, essentially assuming dilute and ideal conditions. We derive the resulting nondimensional combined heat and mass transport model for a ternary solution and present numerical solutions. We include an analysis of the effects of varying some nondimensional parameters on rates of ice growth with comments on the necessity of models that account for spatially varying quantities in cryobiology.

Diagnostic accuracy of transbronchial lung cryobiopsy for interstitial lung disease diagnosis (COLDICE): a prospective, comparative study.

BACKGROUND: Transbronchial lung cryobiopsy (TBLC) is a novel technique for sampling lung tissue for interstitial lung disease diagnosis. The aim of this study was to establish the diagnostic accuracy of TBLC compared with surgical lung biopsy (SLB), in the context of increasing use of TBLC in clinical practice as a less invasive biopsy technique. METHODS: COLDICE was a prospective, multicentre, diagnostic accuracy study investigating diagnostic agreement between TBLC and SLB, across nine Australian tertiary hospitals. Patients with interstitial lung disease aged between 18 and 80 years were eligible for inclusion if they required histopathological evaluation to aid diagnosis, after detailed baseline evaluation. After screening at a centralised multidisciplinary discussion (MDD), patients with interstitial lung disease referred for lung biopsy underwent sequential TBLC and SLB under one anaesthetic. Each tissue sample was assigned a number between 1 and 130, allocated in a computer-generated random sequence. Encoded biopsy samples were then analysed by masked pathologists. At subsequent MDD, de-identified cases were discussed twice with either TBLC or SLB along with clinical and radiological data, in random non-consecutive order. Co-primary endpoints were agreement of histopathological features in TBLC and SLB for patterns of definite or probable usual interstitial pneumonia, indeterminate for usual interstitial pneumonia, and alternative diagnosis; and for agreement of consensus clinical diagnosis using TBLC and SLB at MDD. Concordance and κ values were calculated for each primary endpoint. This study is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12615000718549. FINDINGS: Between March 15, 2016, and April 15, 2019, we enrolled 65 patients (31 [48%] men, 34 [52%] women; mean age 66·1 years [SD 9·3]; forced vital capacity 83·7% [SD 14·2]; diffusing capacity for carbon monoxide 63·4% [SD 12·8]). TBLC (7·1 mm, SD 1·9) and SLB (46·5 mm, 14·9) samples were each taken from two separate ipsilateral lobes. Histopathological agreement between TBLC and SLB was 70·8% (weighted κ 0·70, 95% CI 0·55-0·86); diagnostic agreement at MDD was 76·9% (κ 0·62, 0·47-0·78). For TBLC with high or definite diagnostic confidence at MDD (39 [60%] of 65 cases), 37 (95%) were concordant with SLB diagnoses. In the 26 (40%) of 65 cases with low-confidence or unclassifiable TBLC diagnoses, SLB reclassified six (23%) to alternative high-confidence or definite MDD diagnoses. Mild-moderate airway bleeding occurred in 14 (22%) patients due to TBLC. The 90-day mortality was 2% (one of 65 patients), following acute exacerbation of idiopathic pulmonary fibrosis. INTERPRETATION: High levels of agreement between TBLC and SLB for both histopathological interpretation and MDD diagnoses were shown. The TBLC MDD diagnoses made with high confidence were particularly reliable, showing excellent concordance with SLB MDD diagnoses. These data support the clinical utility of TBLC in interstitial lung disease diagnostic algorithms. Further studies investigating the safety profile of TBLC are needed. FUNDING: University of Sydney, Hunter Medical Research Institute, Erbe Elektromedizin, Medtronic, Cook Medical, Rymed, Karl-Storz, Zeiss, and Olympus.

Cloning, characterization and expression analysis of glutathione S-transferase from the Antarctic yeast Rhodotorula mucilaginosa AN5.

The gene for glutathione S-transferase (GST) in Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 was cloned and expressed in Escherichia coli and named RmGST. Sequence analysis showed that the RmGST gene contained a 843 bp open reading frame, which encoded 280 amino acid residues with a calculated molecular mass of 30.4 kDa and isoelectric point of 5.40. RmGST has the typical C- and N-terminal double domains of glutathione S-transferase. Recombinant RmGST (rRmGST) was expressed in E. coli to produce heterologous protein that had a high specific activity of 60.2 U/mg after purification. The apparent Km values of rRmGST for glutathione and 1-chloro-2,4-dinitrobenzene were 0.35 mM and 0.40 mM, respectively. Optimum enzyme activity was measured at 35 °C and at pH 7.0 and complete inactivation was observed after incubation at 55 °C for 60 min rRmGST tolerated high salt concentrations (1.0 M NaCl) and was stable at pH 3.0. Additionally, the recombinant protein nearly kept whole activity in Hg2+ and Mn2+, and could tolerate Ca2+, Cu2+, Mg2+, Cd2+, EDTA, thiourea, urea, Tween-80, H2O2 and Triton X-100. Real-time quantitative PCR showed that relative expression of the GST gene was significantly increased under Cu2+ and low temperature stress. These results indicate that rRmGST is a typical low thermostable enzyme, while its other characteristics, heavy metal and low temperature tolerance, might be related to its Antarctic home environment.

Effects of malignancies on fertility preservation outcomes and relevant cryobiological advances.

A decrease in cancer deaths has resulted in the possibility of child bearing for many young adult cancer survivors. Most antitumor treatment modalities are detrimental to female fertility, and methods for fertility preservation before gonadotoxic treatment, including cryopreservation of oocytes, embryos and ovarian tissue, have therefore been developed. This review focuses on the ovarian function of cancer patients, the safety and efficacy of fertility preservation methods, and the pregnancy outcomes of these patients. Breast cancer and hematological tumors constitute the majority of cancers in reproductive-aged female oncology patients. Ovarian function may not be impacted by breast cancer cells, while in patients with hematological malignancies, decreases in anti-Müllerian hormone and antral follicle counts have been demonstrated. In most cases, patients can undergo ovarian stimulation without delaying treatment, and a new stimulation protocol known as dual stimulation, which may be more efficient, has now been developed. Birth outcomes are also acceptable in cancer patients.

Using natural honey as an anti-oxidant and thermodynamically efficient cryoprotectant in embryo vitrification.

Embryo cryopreservation is a common practice in reproductive biology and infertility treatments. Despite major improvements over years, the cryoprotectant solutions are still a major source of concern, mostly due to their chemical toxicity and suboptimal protection against cryoinjuries. In this work, we introduced natural honey as a non-permeating cryoprotectant to replace traditionally used sucrose in embryo vitrification. The proposed media were compared with conventional ones by evaluating vitrified/warmed mouse embryos based on their re-expansion, hatching rate and transcription pattern of selected genes involved in heat-shock response, apoptosis and oxidative stress. Despite the similar high re-expansion rate, molecular fingerprint of the cryopreservation is remarkably reduced when honey is used instead of sucrose. The biological response of the proposed media was explained from a fundamental point of view using antioxidant analysis, DSC and GC techniques. It was found that the proposed honey-based medium is less thermodynamically prone to ice formation, which along with its antioxidant capacity can control the production of oxygen radicals and minimize the stress-induced transcriptional response. Furthermore, this work tries to correlate the physico-chemical properties of the vitrification solutions with the cellular and molecular aspects of the cryopreservation and proposes the application of natural cryoprotectants in cryobiology.

Foundations of modeling in cryobiology-II: Heat and mass transport in bulk and at cell membrane and ice-liquid interfaces.

Modeling coupled heat and mass transport in biological systems is critical to the understanding of cryobiology. In Part I of this series we derived the transport equation and presented a general thermodynamic derivation of the critical components needed to use the transport equation in cryobiology. Here we refine to more cryobiologically relevant instances of a double free-boundary problem with multiple species. In particular, we present the derivation of appropriate mass and heat transport constitutive equations for a system consisting of a cell or tissue with a free external boundary, surrounded by liquid media with an encroaching free solidification front. This model consists of two parts-namely, transport in the "bulk phases" away from boundaries, and interfacial transport. Here we derive the bulk and interfacial mass, energy, and momentum balance equations and present a simplification of transport within membranes to jump conditions across them. We establish the governing equations for this cell/liquid/solid system whose solution in the case of a ternary mixture is explored in Part III of this series.