Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed.hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates.A cross-industry study was performed to collect hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used.A systematic approach to estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.This methodology can estimate  •hERG potency/safety margin is a widely used nonclinical cardiac safety strategy.•A new regulatory paradigm promotes the integration of nonclinical and clinical data.•Lack of uncertainty quantification hindered using hERG potency in the new paradigm.•A systematic method was established to address this limitation.•Analysis supports using different safety margin thresholds in different context. Block o reserve , and in  reserve . Althougrization . Based orization . The prorization .IC50, for hERG inhibition to the maximum free therapeutic concentration, Cmaxfree, of the drug. The assumption is that above the safety margin threshold, the compound is not likely to induce TdP  to accommodate for those drugs with solubility issue. Based on the estimated posterior distributions of hERG potency of the 28 drugs, the relationship between choosing a specific safety margin threshold and making an error in TdP risk classification  is explored. Finally, a modeling and simulation study was used to highlight the fact that any hERG block potency estimation depends on the experimental temperature and the particular voltage protocol used.Under the CiPA Initiative, a systematic strategy was developed to address the above issues. A standardized voltage protocol was used in measurements of 28 CiPA drugs with known clinical TdP risk at several globally-distributed facilities (“sites”). All sites used high throughput (HTS) automated patch-clamp systems, though not necessarily the same type of devices. The data were generated in a blinded fashion by each site, and then collected for centralized de-blinding and data analysis. A rigorous statistical method was applied to account for inter- and intra- site variability. A method was also developed to calculate the block potency using not only 22.1hERG data was analyzed here. The identities of the participating sites were masked by numerical indicators. A centralized procedure was taken where all 28 drugs were purchased and prepared into stock solutions by a single laboratory outside of the participating sites. The identities and concentrations of the stock solutions were masked. Each site received a blinded aliquot of the stock solutions. Instructions were provided for making serial dilutions from each stock solution. The sites performed their hERG assays without knowing the identities or concentrations of the compounds. The unblinded drug names and concentrations, along with the measured block percentage for each cell, are available along with this publication at https://github.com/FDA/CiPA/tree/Systematic_Strategy_hERG_Block_2020.The CiPA Initiative organized a panel of facilities with HTS systems to participate in a multi-site study coordinated by the Health and Environmental Sciences Institute (HESI). The study was conducted in two phases  and not all sites participated in both phases. The 28 drugs were categorized into High, Intermediate, or Low TdP risk classes by a dedicated CiPA team . While tThe sources of the 28 CiPA drugs are as follows. For Phase 1, all drugs were purchased from Millipore Sigma , formulated in 100% DMSO, blinded and shipped frozen to collaborator testing sites. All stocks were prepared at 1000× concentration to be tested, to limit final DMSO exposure upon preparation of serial dilutions to 0.1% in each assay. For Phase 2, blinded drug powder with instruction for formulation of stock solutions was sent to the sites by the Chemotherapeutic Agents Repository of the National Cancer Institute and stored at −20 °C until the day of testing.The CiPA step-ramp protocol was used as the standard protocol across sites. It involves a pulse pattern, repeated every 5 s, consisting of a depolarization to 40 mV amplitude for a 500 ms duration, followed by a ramp (1.2 V/s) to −80 mV for 100 ms. The holding potential is −80 mV. Peak tail current is measured during the ramp. Site-specific parameters, such as cell lines, buffers, HTS platforms, can be found in Supplementary Materials. The details of the action potential waveform protocol can be found in a previous publication .2.2IC50 for the jth site (denoted by pIC50j) across all sites was assumed to follow a logistic distribution. Similarly, the site-specific Hill coefficients (hj) across all sites were assumed to follow a log-logistic distribution. The hyperparameters controlling these distributions (inter-site variability), along with site-specific probability distributions of IC50 (and Hill coefficients) within each site (intra-site variability), are estimated through a Markov Chain Monte Carlo (MCMC) process using the Metropolis-Hastings algorithm. In-house developed R scripts using the FME package (https://cran.r-project.org/web/packages/FME/index.html) were run on the FDA's High Performance Computing cluster to execute the algorithms. A discussion of the prior information, bounds on the parameters, and implementation details can be found in the Supplementary Methods.We used a Bayesian hierarchical model (BHM) similar to that used by Johnstone et al. for analysis of ion channel dose-response data . The dis2.3hERG dynamic model previously developed for CiPA . This is similar to a modified Hill equation used to introduce an Imax effect. Details can be found in the Supplementary Methods and in reference . Theoretically, intra-site variability may be mainly attributed to the inherent randomness of the measurement  while inter-site variability may largely stem from systematic differences between sites . To account for both types of variability, we adopted a Bayesian hierarchical modeling (BHM) approach  IC50s and Hill coefficients across sites can be modeled as a “higher level” distribution to quantify inter-site variability. Within each individual site, the system-specific IC50 and Hill coefficient distributions for each drug determine a “lower level” uncertainty in dose-response relationships that characterize intra-site variability. The hyperparameters that describe inter-site variability and the site-specific parameters that describe intra-site variability can be estimated from the multi-site data, as shown as a diagram in μ (see Supplementary Methods), which corresponds to the mean of the estimated 50IC distribution across sites for each drug, is shown in An initial examination of the multi-site approach . The genhERG channel often cannot attain close to 50% block in experimental practice. This makes IC50 for such drugs difficult to estimate. To remedy this problem, IC20 has been proposed as a substitute for IC50 to quantify hERG block potency  after taking into consideration the experimental variability  without considering a desired high specificity . We reasoned that different context of use might place different weights on the tolerability of false positives and false negatives, which would motivate using different thresholds. Accordingly, the relationship between any chosen safety margin threshold and the rates of false positive (probability of low TdP risk drugs having the safety margin below the threshold) and false negative (probability of high or intermediate TdP risk drugs having the safety margin above the threshold) is explored based on the posterior probability distributions of the 28 drugs' hERG potency divided by their Cmaxfree  to defiCmaxfree  values  (One major confounding factor in the  results . But no rameters . By usinrameters . As the (0.2 Hz) , a slowe(0.2 Hz) . Before IC50s across the three protocols for all CiPA drugs is shown in hERG channel during protocol intervals (e.g. bepridil (IC50s when varying the frequency from 0.2 Hz to 0.03 Hz. Some highly trapped drugs (e.g. dofetilide (IC50) at slower (0.03 Hz) compared to higher (0.2 Hz) frequency for the same CiPA protocol, illustrating the so called reverse frequency dependent blocking as previously reported (hERG block potency is higher (the IC50 is lower) at higher frequency. Other than the tendency of trapping, drug binding rate also plays a role. For instance, terfenadine and ondansetron are two drugs with similar tendency of trapping, and yet quite different binding rates (IC50s) when the CiPA step-ramp protocol frequency is decreased from 0.2 Hz to 0.03 Hz. In contrast the fast-binder ondansetron has almost the same level of block potency when frequencies change. These results show that hERG potency estimates depend strongly on the applied voltage protocol. Therefore, any proposed safety margin only has scientific validity when it is in reference to a specific voltage protocol.A comparison of the predicted bepridil ) have esfetilide ) even hareported . By contreported , have frng rates . The slo4hERG block potency for TdP liability assessment. This strategy aims at addressing four issues associated with previous studies: the use of heterogenous experimental conditions (voltage protocols and temperatures), the ambiguity in the endpoint (TdP vs QTc prolongation), the lack of uncertainty quantification, and the inability to cover those drugs that are difficult to estimate IC50s due to various reasons such as solubility.In this study, we developed a systematic approach to estimate hERG assays at each site. The collected multi-site data are an important resource to investigate “why” experimental variabilities exist and identify the most important underlying factors, with the goal of reducing lab-to-lab variability for future hERG assays. On the other hand, the collected data also provide a resource to study “how” to use current hERG assay data across industry, for example to define a safety margin threshold after considering intra- and inter- site variability. While a manuscript for the former is being prepared, this document represents our effort towards the latter application of the data.The standardization of experimental protocols was performed as part of a cross-industry HTS automated patch clamp study coordinated by HESI under the CiPA Initiative. The step-ramp protocol was chosen as it is a simple approximation of the shape of a cardiac action potential. Some physiological temperature data were also collected by the HESI study. As most of the data were at ambient temperature, we decided to focus on room temperature data in this project to maximize the coverage of participating sites for a more comprehensive understanding of inter-site variability. To make all sites' experimental conditions as close as possible, not only the voltage protocol but also the sources and concentrations of each compound are “standardized”  across sites. A blinding procedure was also implemented to ensure an objective application of the hERG block potency using low inhibitory concentrations (IC20 etc.) to accommodate those drugs with poor solubility or not enough block at highest concentrations. It should be noted that it may be difficult to calculate a reasonable estimate of ICx if the drug in experimental practice cannot achieve a block of at least x. For example, to calculate IC20 accurately the drug will need to achieve at least 20% block in the hERG assay at highest tested concentrations.The Bayesian hierarchical modeling approach allows us to quantify intra-site and inter-site variability in a sound statistical framework. A supplementary approach was also developed to define hERG potency (IC50 or IC20) values as for the 28 drugs derived from BHM, we explored the relationship between safety margin threshold, the false positive rate, and the false negative rate for predicting TdP risk. Similar to previous studies  could be applied to determine which drugs have low probability of hERG block-mediated TdP liability, and which ones may have enough concern and warrant the use of more comprehensive models for accurate TdP risk assessment.Based on the posterior probability distributions of  studies  we found studies . This ishERG block potency in our multi-site study, no uncertainty was associated with Cmaxfree when calculating the safety margin. This is due to the fact that there are very few studies to establish methods to quantify the uncertainty in the estimation of plasma protein binding, a main factor behind the variability of Cmaxfree . Although reverse use dependency on AP prolongation is often observed at the cellular level for many hERG blockers . And while all sites used heterologous cell lines to express human hERG channels, different cell types (CHO or HEK293) and biological properties (e.g. channel density) might also contribute to cross-site variability. Our BHM method quantifies the overall inter-lab variabilities from all sources and does not require any specific experimental conditions to be used by different labs (such as heterologous vs iPS or native cardiomyocytes), as long as the underlying experimental protocol is appropriately designed for block potency estimation and standardized across labs. However, understanding the impact of these experimental conditions on potency estimation is essential for future hERG assays.A second limitation is that we used model-predicted, rather than experimentally measured, blockers , at the blockers . Reverseew drugs . It is uhERG potency estimation and re-define the safety margin.With the proposed establishment of best practice and quality standards for in vitro ion channel studies by the ICH S7B/E14 Q&A process , it is hThis report is not an official US Food and Drug Administration guidance or policy statement. No official support or endorsement by the US Food and Drug Administration is intended or should be inferred.The authors declared no conflict of interests.


Insulin resistance increases patients’ risk of developing type 2 diabetes (T2D), non-alcoholic steatohepatitis (NASH) and a host of other comorbidities including cardiovascular disease and cancer. At the molecular level, insulin exerts its function through the insulin receptor (IR), a transmembrane receptor tyrosine kinase. Data from human genetic studies have shown that Grb14 functions as a negative modulator of IR activity, and the germline Grb14-knockout (KO) mice have improved insulin signaling in liver and skeletal muscle. Here, we show that Grb14 knockdown in liver, white adipose tissues, and heart with an AAV-shRNA (Grb14-shRNA) improves glucose homeostasis in diet-induced obese (DIO) mice. A previous report has shown that germline deletion of Grb14 in mice results in cardiac hypertrophy and impaired systolic function, which could severely limit the therapeutic potential of targeting Grb14. In this report, we demonstrate that there are no significant changes in cardiac function as measured by echocardiography in the Grb14-knockdown mice fed a high-fat diet for a period of four months. While additional studies are needed to further confirm the efficacy and to de-risk potential negative cardiac effects in preclinical models, our data support the therapeutic strategy of inhibiting Grb14 to treat diabetes and related conditions. It is the primary anabolic hormone in the body which regulates the metabolism of carbohydrates, lipids and proteins. It promotes the absorption of glucose into liver, fat and muscle cells. Insulin resistance occurs when the cells fail to efficiently absorb glucose in response to insulin stimulation. It underlies T2D, NASH and a host of other comorbidities including cardiovascular disease and cancer2. It is an intracellular adaptor protein that contains multiple domains including a proline-rich region, a Ras-associating (RA) domain, a pleckstrin homology (PH) domain, a between the PH and SH2 (BPS) domain, and a Src homology 2 (SH2) domain. Grb14 binds to receptors and non-receptor tyrosine kinases and regulates a variety of signaling pathways2. Multiple genome wide association studies have found that loci near Grb14 are significantly associated with waist to hip ratio, blood lipids, body fat percentage and fasting insulin levels in humans, indicating that Grb14 plays a role in regulating metabolism7.Grb14 is a member of the growth factor receptor-bound protein family9. It has been reported that Grb14 binds to the insulin receptor (IR) and negatively regulates the catalytic activity of IR11. A crystal structure shows that Grb14 binds to IR in a pseudo-substrate inhibitor fashion11. The physiological relevance of the regulation of IR by Grb14 was confirmed in vivo in the Grb14-KO mice8. The whole-body Grb14 deficient mice exhibited improved glucose homeostasis and enhanced insulin signaling. However, the mice exhibited cardiac hypertrophy and impaired cardiac function as measured by echocardiography12. This finding raises safety concerns on inhibiting Grb14 as a therapeutic strategy to treat insulin resistance and its related diseases. In this study, we have examined the effects of Grb14 knockdown on metabolism and cardiac function in diet-induced obese (DIO) mice with an AAV carrying shRNA against Grb14 (Grb14-shRNA). We show that Grb14-shRNA results in efficient knockdown of Grb14 in the liver, the white adipose tissue and the heart, and that it improves glucose homeostasis without causing adverse effects on cardiac function in DIO mice.Grb14 is primarily expressed in metabolically active tissues including liver, fat and skeletal muscle10. To determine if this regulation is relevant in humans, we analyzed the expression of the GRB14 gene in human adipose tissues  and an AAV carrying the empty vector as control. To determine the knockdown efficiency of the Grb14-shRNA, we injected AAVs to the normal chow-fed mice via tail vein and collected the liver, epididymal white adipose tissue (eWAT), skeletal muscle, and heart four weeks after injection. Grb14 gene expression was decreased by ~90% in the liver and ~80% in the heart and in the white adipose tissue in the mice treated with Grb14-shRNA compared to the empty vector control Fig. . To asseG6Pc) was down-regulated significantly with Grb14-shRNA treatment  and diacylglycerol O-acyltransferase 2 (Dgat2), two lipogenic genes, were also decreased with Grb14-shRNA treatment  remain the only class of FDA-approved insulin sensitizers. The undesired side effects associated with the use of TZDs include weight gain, edema and bone fracture17. Therefore, there is a large unmet medical need for effective and safe insulin sensitizers.It is estimated that over one third of adults in the US have insulin resistance18. Furthermore, our data demonstrate that there is a negative correlation between Grb14 gene expression in adipose tissue and metabolic health in humans , anti-sense oligonucleotide (ASO) or siRNA.Protein-protein interaction (PPI) is critical in signal transduction pathways. The pharmaceutical industry has been pursuing modulators that either enhance or disrupt PPI for drug development for decades23. We also found that Grb14-shRNA reduced expression of the lipogenic genes Scd1 and Dgat2 in the liver . All subjects completed a screening medical evaluation that included a history and physical examination, a resting electrocardiogram, and standard blood tests. All studies were approved by the Institutional Review Board of Washington University School of Medicine in St. Louis, MO, and written informed consent was obtained from all subjects before their participation. In addition, all experiments were performed in accordance with relevant guidelines and regulations.26.Study 1 was a cross-sectional analysis of participants with metabolically healthy obesity , defined as BMI 30–50 kg/m2 and fasting plasma glucose concentration <100 mg/dl, 2-h OGTT plasma glucose concentration <140 mg/dl, and HbA1c ≤ 5.6%, or metabolically unhealthy obesity (MUO) , defined as BMI 30–50 kg/m2 and fasting plasma glucose concentration ≥100 mg/dl, or 2-h OGTT plasma glucose concentration ≥140 mg/dl, or HbA1C ≥ 5.7%. Study 2 was a longitudinal analysis of women with obesity before and after 20 ± 1% weight loss, induced by lifestyle therapy  or Roux-en-Y gastric bypass surgery . Study 3 was a longitudinal analysis of people with MUO before and after 5.9 ± 0.3% weight gain, induced by consuming an additional 1000 kcal/day until a targeted 5–7% weight gain was achieved . The effects of weight loss and weight gain on body composition, insulin sensitivity and expression of other adipose tissue genes have been reported previously2 body surface area (BSA)/min (initiated with a two-step priming dose of 200 mU/m2 BSA/min for 5 min followed by 100 mU/m2 BSA/min for 5 min). Euglycemia (~100 mg/dL) was maintained by variable rate infusion of 20% dextrose. Blood samples were obtained before beginning the hyperinsulinemic-euglycemic clamp procedure to determine basal, postabsorptive plasma glucose and insulin concentrations. Biopsies from abdominal subcutaneous adipose tissue were obtained before starting insulin and dextrose infusion while subjects laid at rest in bed. The periumbilical area was cleaned and anesthetized with 1% lidocaine injection, and a small skin incision (~0.5 cm) was made. Adipose tissue was then aspirated through a 4-mm liposuction cannula  connected to a 60-cc syringe. Samples were immediately rinsed in ice-cold saline and frozen in liquid nitrogen before being stored at −80 °C until further processing.After subjects fasted for 12 h overnight, a hyperinsulinemic-euglycemic clamp procedure was conducted to assess whole-body insulin sensitivity. An intravenous catheter was inserted into a forearm vein to infuse insulin and dextrose and an additional catheter was inserted into a radial artery to obtain arterial blood samples. Insulin was infused at a rate of 50 mU/mPlasma glucose concentration was measured by an automated glucose analyzer (Yellow Springs Instruments Co.). Plasma insulin concentration was measured by using electrochemiluminescence technology . Total RNA was isolated from frozen subcutaneous adipose tissue samples by using QIAzol and RNeasy mini kit (QIAGEN). Library preparation was performed with total RNA and cDNA fragments were sequenced on an Illumina HiSeq-4000. The fragments per kilobase million reads (FPKM) values were calculated and used for further gene expression analyses. The RNA-seq data have been deposited in the NCBI’s Gene Expression Omnibus database (GEO GSE143319).27. Briefly, ten short hairpin RNA (sh1-sh10) of 19 nucleotides, with the starting position at nucleotide 536, 437, 294, 321, 1577, 660, 1320, 485, 1211, 1547 (NM_016719) respectively, were designed to specifically target mouse Grb14. Forward and reverse oligonucleotides were annealed and ligated into the BbsI site of AAV cis plasmid containing the human H1 promoter to drive shRNA expression. To screen for an shRNA that efficiently inhibit mouse Grb14, we transfected the ten candidate AAV-shRNAs into Hepa1c1c7 cells that express mouse Grb14 endogenously using 4D-Nucleofector  per the manufacturer’s instruction. Cells were harvested 48 hours post transfection, and RNA was isolated and prepared for RT-PCR analysis. The most potent shRNA (sh6) was selected for AAV9 packaging. AAV9-H1-Grb14sh6 and the negative control AAV vector that contains the H1 promoter and termination sequence were produced by the helper-free triple-plasmid transfection method at University of Massachusetts Medical School Viral Vector Core. The AAV cis plasmid, an adenoviral helper plasmid and a chimeric packaging plasmid containing the AAV2 Rep gene and AAV9 Cap gene were co-transfected into HEK293 cells. AAV vectors were subsequently purified by two rounds of cesium chloride density gradient ultracentrifugation, and titers were determined via RT-PCR analysis.To generate AAV encoding potent short hairpin RNA (shRNA) against mouse Grb 14, we followed a previously described protocol11 viral genome copies/mouse) via tail vein and immediately subjected to the HFD challenge. All experiments were approved by the Institutional Animal Care and Research Advisory Committee of Merck & Co., Inc., South San Francisco, CA, USA and were performed in accordance with relevant guidelines and regulations.Male C57BL/6J mice were purchased from the Jackson Laboratory. Mice were fed with standard chow or an HFD containing 60% fat-derived calories  ad libitum. For AAV treatments, mice were randomized based on body weight and ad lib blood glucose levels. 10-week old mice were injected with AAV  via oral gavage at the dose of 2 g/kg body weight. Blood glucose was measured via tail bleeding at 15, 30, 60, and 120 minutes post glucose administration.Blood was collected via tail bleeding into Microvette tubes coated with EDTA  and spun at 8,000 rpm for ten minutes in a bench-top centrifuge at 4 °C to separate plasma. Insulin levels were measured using a commercial insulin ELISA kit  according to the manufacturer’s instruction.28.Total RNA was extracted from tissues with RNeasy mini kits (Qiagen). Two micrograms of RNA from each sample were then used to generate cDNA with SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen). All TaqMan probes were purchased from Life Technologies. QPCR was run on Applied Biosystems ViiA 7 system. The data were analyzed using the delta-delta Ct method by using PPiB as the internal controlIn Vivo Imaging System , at heart rates >350 bpm. Electrocardiogram and heart rate were monitored throughout the imaging procedure. Echocardiographic measurements were taken from the two-dimensional short axis (m-mode) recordings of the LV. For each parameter, three images from consecutive cardiac cycles were measured and averaged. One reader analyzed all the echocardiograms in a blinded manner.Echocardiograms was acquired by short axis M-mode under spontaneous respiration with 1–2% isoflurane in an oxygen mix. Images were acquired with the use of a Vevo 2100™ High-Resolution The sequence corresponding to the insulin receptor isoform B was Gibson cloned into the LgBiT C-terminal fusion plasmid (Promega cat#N2014), the sequence corresponding to Grb14 was Gibson cloned into the C terminal SmBiT fusion plasmid (Promega cat#N2014) using standard protocols. Construct accuracy was confirmed by Sanger sequencing. HEK293T cells (ATCC cat#CRL-3216) were maintained in DMEM (Gibco cat#11995-065) plus 10% FBS (Gibco cat#26140-079) at 37 °C and 5% CO2.For the split NanoLuc assay 4,000 HEK 293T cells were plated per well in a TC treated white opaque 96 well plate (Corning cat#3917). The plate was placed in the incubator and the cells were allowed to adhere overnight. The following day the cells were transfected with the InsR-LgBiT and Grb14-SmBiT or in a 2.5:1 ratio using Fugene HD (Promega cat#E2311) according to the manufacturer’s instructions and the plate was returned to the incubator. 24 hours later the media was changed to 100 µL of DMEM without serum and the cells were placed back in the incubator. 16 hours later the media was exchanged for fresh serum starve media. 2 h later the Nano-BiT protocol was run using a dose response of insulin according to the manufacturers protocol. Briefly, the plate was equilibrated to room temperature for 10 min. 25 µL of the NanoBit substrate and buffer (Promega Cat#N2014) is added to each well and the luminescence is read for 5 min on the EnVision (PerkinElmer). At the completion of the 5 min read 10 µL of insulin or PBS diluted in DMEM with no serum to provide the indicated final concentration of insulin was added to each well and luminescence was read every 1.5 minutes for 30 minutes. The luminescence signal after the addition of insulin was normalized to the luminescence signal for that individual well at the end of the 5-minute pre-insulin read.6 cells/well in a 24-well SureCoat-treated plate. Small interference RNA (siRNA) was purchased from Thermo Fisher Scientific and transfection was performed according to the manufacturer’s instruction.Neonatal rat cardiomyocytes were isolated from the hearts of 14 neonatal SD rats using Cellutron Life Technology (NC 6031) kit following the manufacturer’s instruction. All the solutions  were diluted and prepared per the manufacturer’s protocol. Briefly the 24-well plates were coated with pre-warmed SureCoat solution until further use. The neonatal rats were sterilized with 70% ethanol before and after decapitation. The chest was opened with sharp scissors and the heart was quickly removed and placed in 50 mL falcon tube containing ice-cold D1 buffer. The hearts were washed twice with ice-cold D1 buffer. The buffer was carefully removed, and the hearts were transferred to a 25 mL beaker containing a magnetic stir bar. The digestion buffer (D2 + EC) was added to the beaker and the beaker was placed on a magnetic stirrer at 37 °C (~150 rpm) and incubated for 12 min. The mixture was pipetted a few times after the solution started becoming cloudy due to collagenase activity. The cloudy supernatant was then transferred to a 15-mL falcon tube and centrifuged at 300 × g for 2 min at room temperature. The supernatant was removed and the pellet was resuspended in 5 mL of D3 solution. The procedure from addition of D2 + EC to the tissue to resuspension of pellet containing the cells in D3 solution was repeated until all the tissue was digested by collagenase (4×−5×). All the resuspended solution was collected in a falcon tube and was filtered through cell strainer. Finally, the cells were counted and pre-plated for 45 min in an uncoated plate and incubated at 37 °C, 5% CO2 to reduce contamination from cardiac fibroblasts. After 45 min, the unattached cells were carefully removed and gently washed with 1X NS medium. The cells were then centrifuged at 300 × g and the pellet was resuspended in NS medium and plated at ~1 × 10Statistical analyses were conducted with Prism 8.1.1 . Comparisons between multiple groups across multiple time points were analyzed using two-way analysis of variance (ANOVA), followed by Turkey’s multiple comparison test. Comparison of heart weight between groups was analyzed using one-way ANOVA. Comparisons between two groups were analyzed using Student’s t-test. Statistical significance was defined as P < 0.05.Supplementary Data.


Bacillus megaterium 1259, as a novel bacterium, has been used as a probiotic in poultry feed. In this study, we analyzed the genome sequence and genetic characteristics of Bacillus megaterium 1259 in order to have a deep understanding of its genomics information. Moreover, the effects of Bacillus megaterium 1259 as a probiotic on lactating dairy cow performance were determined due to the different digestive systems of dairy cows from poultry. The results suggest that Bacillus megaterium 1259 can increase milk production and will not bring any side effects on blood metabolites in dairy cows.Probiotics play a vital role in animal production. Bacillus megaterium 1259 (BM1259), from chicken manure. Whole-genome sequencing analysis showed that the BM1259 complete genome is composed of a 5,043,095 bp circular chromosome and three circular plasmids, and it encodes 5379 coding genes and 182 RNA genes. Among these genes, a series of nitrate assimilation-related genes and pathways were identified, implying a potential role of BM1259 in nitrate metabolism. In addition, 24 lactating Holstein dairy cows were randomly assigned to four groups that were fed a total mixed ration (TMR) diet only (C), a TMR diet supplemented with 5 g/day of BM1259 (T1), a TMR diet supplemented with 10 g/day of BM1259 (T2), or a TMR diet supplemented with 15 g/day of BM1259 (T3). The results showed that supplementing dairy cows with 15 g/day of BM1259 increased 4% fat-corrected milk production. The molar proportion of propionate (C3) was significantly higher in T2 than in C. The C2:C3 ratio of T3 was higher than those of C and T2. No negative effect of BM1259 on blood indicators was detected. This study demonstrates BM1259 can be applied as a potential probiotic to improve nitrogen utilization and milk production in lactating dairy cows.In this study, we isolated a novel bacterium,  Bacillus, spore-forming bacteria, are becoming increasingly popular as probiotics used in animal feed. For example, Bacillus spp. strains were analyzed for usage as probiotic additives in pig feed [Bacillus sp. probiotics may promote subsequent milk yields and fat and protein contents [Probiotics are live microorganisms that bring health benefits to the host when administered in adequate amounts . They plpig feed . Kritas contents .Bacillus subtilis natto, as a probiotic, has positive effects on rumen fermentation, the ruminal microbiome, and milk production in dairy cows [Bacillus sp., Bacillus licheniformis, improved ruminal apparent nutrient digestibility of organic matter, neutral detergent fiber, and acid detergent fiber in Holstein cows [Studying the effects of a certain microbial strain on various animal species, growth conditions, and diet types may help to clarify the conditions under which probiotics could work in animal production. In dairy cow production, iry cows . Anotherein cows .Bacillus megaterium is a gram-positive, aerobic spore-forming neutralophilic bacterium that is ubiquitous in various environments but commonly survives in the soil. It has a potential role as an industrial probiotic due to its adaptability to a wide temperature environment (3–45 °C) and utilization of different carbon sources [B. megaterium has the potential to be applied as a probiotic in the feed industry [B. megaterium 1259 (BM1259) was isolated from chicken manure, and fermentation technology for producing it has been well developed. In our previous studies, we found that diets supplemented with BM1259 could improve the performance of laying hens and decrease the ammonia nitrogen emissions of excrement [ sources ,12. To d sources ,14. Althindustry ,16, furtxcrement . To dateGiven the dearth of genetic information, we analyzed the genome sequence and genetic characteristics of BM1259, which might be used as a new feed additive for animal production. The second objective of this study was to investigate the effects of BM1259 on lactating dairy cow performance. This work will provide genomic information for a deep understanding of BM1259 and provide a new bacterium strain for the probiotic industry for dairy cow production.The bacterial strain BM1259 was isolated from chicken manure in Yangzhou city and deposited in the China General Microbiological Culture Collection Center (CGMCC No 1259). This strain was identified based on the 16S ribosomal DNA sequence by multiple alignment and phylogenetic analysis and cultured in our own laboratory A in thishttp://circos.ca) [The genome sequence data were deposited in NCBI GenBank, and the accession number is PRJNA506612. The complete genomic DNA of BM1259 was obtained by a QIAGEN Genomic DNA extraction kit , according to the standard manufacturer methods, and quantified using a NanoDrop One UV-Vis spectrophotometer . A nanopore sequencing library of genomic DNA was prepared at Nanjing Aurora Gene Technology Co., Ltd., China. All long DNA fragments from extracted DNA samples were selected using the BluePippin system . The ends of all long DNA fragments were attached with adapters supplied by the Ligation Sequencing kit . A Qubit 3.0 Fluorometer  was used to evaluate the size of library fragments. Finally, the DNA library was added into a flow cell, and sequencing was performed on the GridION sequencing platform. The raw signal data were translated into raw DNA reads using base calling. Overlap–layout–consensus algorithms were used for the assembly of nanopore sequencing reads to generate an overlap graph. Then, a draft assembly was constructed according to previous methods . The assrcos.ca) . Based ohttp://www.megasoftware.net/), as described in a previous report [Based on the 16S rRNA gene from the BM1259 genome, the full-length 16S rRNA sequence was amplified using PrimeSTAR Max DNA Polymerase kits . Sequences encoding 16S rRNA of BM1259 and other bacteria were initially aligned with Clustal W and displayed using GeneDoc software. Phylogenetic trees were analyzed using the maximum likelihood method in MEGA 6 . All procedures were conducted in the animal experiment were managed in accordance with the laws and regulations approved by the animal welfare office of Yangzhou University (YZUDWLL-201703-001).8 cfu/g); (iii) T2—cows were fed with the TMR diet supplemented with 10 g/day of BM1259 powder (1 × 108 cfu/g); and (iv) T3—cows were fed with the TMR diet supplemented with 15 g/day of BM1259 powder (1 × 108 cfu/g). The experimental period was 11 weeks, consisting of a 2-week adaptation period and a 9-week sample collection period. All cows had free access to water throughout the entire experiment.There were 24 Holstein cows with similar body weights, milk yields, and parities used and divided into four groups with a randomized block design. All cows were housed in individual tie stalls and milked daily at 06:00, 14:00, and 20:00 h. All cows were cultured with a common total mixed ration (TMR) diet  for 7 daThe offered and refused feed amounts were recorded on the third and fourth days every other week throughout the entire experimental period. All samples were dried in an oven at 65 °C for 48 h, ground with a Wiley mill to pass through a 2 mm screen, and then stored for further analyses.Milk yield was recorded each day, and milk samples were collected on the last two days in each week in the experimental period. The 4% fat-corrected milk (FCM) yield was calculated using the following equation (1):Milk samples were composited proportionally  for analyzing the contents . A 50 mLg for 5 min and the supernatants were stored at −20 °C for further analyses. Ruminal volatile fatty acids (VFA) were measured by gas chromatography  equipped with a flame ionization detector.The rumen fluid samples were collected at 4 h after feeding on the morning of the last day of the experimental period. The rumen fluid was filtered through four layers of gauze, and then analyzed for pH immediately by a glass electrode pH meter. It was then centrifuged at 500× g at 4 °C for 10 min to collect serum, which was frozen at −20 °C for further analysis. Serum samples were analyzed for quantification of total protein (TP), albumin (ALB), globulin (GLOB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and uric acid (UA), according to methods described in previous studies [Jugular blood samples of approximately 10 mL were collected from each cow in vacuum plasma tubes 3 h after the morning feeding on the third day of weeks 4, 8, and 12 . The col studies .p < 0.05.All data were analyzed using the MIXED procedure with repeated measurements with the SAS software system . Significance was declared at B. megaterium ATCC 14581 , PM2 , and PM3 . The GC content of the chromosome is 38.25%. Local GC content variation and a clear-cut GC skew at the origin of replication (ori) are evident . A totalB. megaterium strains, the three largest plasmids of BM1259 were also analyzed and predicted in our study  were annotated based on a set of sequential BLAST searches using the GO, KEGG, COG, RefSeq, Pfam, and TIGRFAM databases . COG anaDepending on functional annotation, the genes involved in nitrate metabolism were screened and compared by performing BLASTP against the KEGG database. There were 12 nitrate assimilation-related genes identified from the annotated genes . Among tp < 0.05) but not that of T2  was higher in T2 than in the other treatments. In addition, T3 had a higher C2:C3 ratio than T2. No significant differences were found among the treatments in the ruminal pH, NH3-N content, total VFA concentration, or butyrate content (nC4) . HoweverT1 and T2 had significantly higher TP and GLOB concentrations than C. No differences were observed among treatments in ALB, AST, or UA concentrations . The conB. megaterium, named for its large size (approximately 1.5 × 5 µm), was first discovered by Anton De Bary in 1884 [B. megaterium strains. In recent decades, several features of B. megaterium have been discovered, including sporulation, germination, antibiotic resistance, UV sensitivity [B. megaterium strains, QM B1551 and DSM319, harbor 5,097,129 bp and 5,097,447 bp circular chromosomes, respectively. DSM319 is a natural strain with few plasmids, while QM B1551 contains seven indigenous plasmids, with sizes from 5.4 kb to over 164 kb, transcribing 8 to 176 genes [B. megaterium strains. in 1884 . The newsitivity ,25,26, asitivity ,27,28, aB. megaterium NCT-2 can utilize nitrate as its only nitrogen source for growth and enhance nitrate removal from the soil [Bacteria utilize nitrate as an important source of nitrogen. Bioinformatics analyses of bacterial genomes have revealed structural and regulatory genes responsible for the nitrate assimilation phenotype . Zhou’s the soil ,32. Our Bacillus subtilis can be used as a biocontrol agent to prevent pathogenic microorganisms [Bacillus subtilis natto before weaning could reduce milking time and positively improve calf growth performance [Many reports have demonstrated that probiotics can improve the production of various animals ,34,35,36rganisms ,38 whilerganisms , therebyformance . In our 3-N, and total VFA. Ruminal pH was not affected by treatment in our study, which indicated that BM1259 did not have a negative effect on ruminal pH in lactating dairy cows  may be a potentially useful probiotic in dairy cows, with a recommended dose of 10 or 15 g/day per head. Further research is necessary to uncover the mechanism by which BM1259 improves nitrogen utilization in lactating dairy cows.In summary, the addition of BM1259 to the diets of dairy cows in the lactation period had a positive effect on 4% FCM production. Moreover, supplementation with BM1259 increased C3 in the rumen and reduced NH


A role for the cytoplasmic protein synphilin-1 in regulating energy balance has been demonstrated recently. Expression of synphilin-1 increases ATP levels in cultured cells. However, the mechanism by which synphilin-1 alters cellular energy status is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 on the AMP-activated protein kinase (AMPK) signaling pathway, which may affect energy balance. Overexpression of synphilin-1 increased AMPK phosphorylation (activation). Moreover, synphilin-1 interacted with AMPK by co-immunoprecipitation and GST (glutathione S-transferase) pull-down assays. Knockdown of synphilin-1 reduced AMPK phosphorylation. Overexpression of synphilin-1 also altered AMPK downstream signaling, i.e., a decrease in acetyl CoA carboxylase (ACC) phosphorylation, and an increase in p70S6K phosphorylation. Treatment of compound C (an AMPK inhibitor) reduced synphilin-1 binding with AMPK. In addition, compound C diminished synphilin-1-induced AMPK phosphorylation, and the increase in cellular ATP (adenosine triphosphate) levels. Our results demonstrated that synphilin-1 couples with AMPK, and they exert mutual effects on each other to regulate cellular energy status. These findings not only identify novel cellular actions of synphilin-1, but also provide new insights into the roles of synphilin-1 in regulating energy currency, ATP. Recent studies in transgenic animal models suggest that synphilin-1 is involved in regulating energy homeostasis . SynphilAMP-activated protein kinase  is an integrator of regulatory signals that modulate the systemic and cellular energy status in response to environmental nutritional changes ,23,24,25AMPK and synphilin-1 are both predominantly expressed in the cytosol. However, whether synphilin-1 and AMPK affect one another in controlling energy balance is not known. Thus, in this study, we used molecular biology tools and biochemical approaches to explore the relationship and functional consequences of the interactions between synphilin-1 and AMPK. Our findings identified a novel interaction between synphilin-1 and AMPK, which regulates cellular ATP levels. Therefore, we conclude that synphilin-1 interacts with AMPK and modulates energy homeostasis in cells.Increased AMPKα phosphorylation at threonine 172 reflects increased AMPK activation . To asseMoreover, synphilin-1 also altered AMPK downstream targets, i.e., it decreased acetyl CoA carboxylase (ACC) phosphorylation C,D, and To further investigate the relationship between synphilin-1 and AMPK, we tested whether synphilin-1 interacts with AMPK. Synphilin-1 is a cytoplasmic protein and has been shown to interact with other cytoplasmic proteins ,13,19. WTo further map the interaction regions of synphilin-1 with AMPK, HA-tagged truncated synphilin-1 constructs were transfected into HEK 293 cells. The cell lysates were subjected to anti-HA co-IP, followed by Western blot analysis using anti-AMPK antibody. The F1B (1–246 aa), F1C (1–349 aa), F3 (550–659 aa), F4 (550–769 aa), and F6 (550–919 aa) sites interacted with AMPK . In contOn one hand, reduction of synphilin-1 expression by siRNA significantly attenuated AMPK phosphorylation, compared with cells expressing random control RNA . WhereasATP is the major cellular molecule responsible for providing energy for various cellular processes . AMPK reIn this study, we found that synphilin-1 interacted with AMPK, increased AMPK phosphorylation, and resulted in altered activity of the downstream signaling of ACC and p70S6. Knockdown of synphilin-1 reduced AMPK phosphorylation, while inhibition of AMPK by compound C reduced synphilin-1 and AMPK interactions. Moreover, compound C also reduced the amount of synphilin-1-induced AMPK phosphorylation, and cellular ATP increase. These findings indicate that the interaction of synphilin-1 and AMPK play critical roles in regulating cellular energy balance. To our knowledge, this is the first report showing that synphilin-1 alters AMPK signaling, thereby regulating cellular energy status.Synphilin-1 is a cytoplasmic protein that was cloned over a decade ago . SynphilAMPK regulates systemic and cellular energy status, and functions as an energy sensor to monitor cellular energy status in response to nutritional changes ,22,23,24Our results showed that overexpression of synphilin-1 increased AMPK phosphorylation (activation) compared with levels in vector control cells, while knockdown of synphilin-1 attenuated this increase. Moreover, synphilin-1 action on AMPK altered two major downstream targets of the AMPK pathway (ACC and p70S6K) and increased cellular ATP levels. We found that synphilin-1 decreased ACC phosphorylation, in accord with prior reports, which is involved in ATP production by promoting fatty acid oxidation and preventing fatty acid synthesis ,32. We aSynphilin-1-induced AMPK-mediated increases in cellular ATP levels could contribute to two types of cellular activities. One is to provide ample energy for various cellular functions, such as cell growth and protection against toxic stimuli . PreviouIn summary, these studies reveal novel cellular functions of synphilin-1 in regulating AMPK-linked signaling pathways, thereby contributing to increases in cellular ATP levels. These findings provide a new molecular basis for understanding synphilin-1 biology and the roles of synphilin-1 in regulating energy homeostasis.Media for cell culture, anti-hemagglutinin (HA) polyclonal antibody, and LipofectAMINE Plus reagent were ordered from Invitrogen . Anti-AMPKα (AMPK α-subunit), anti-phosphorylated AMPKα (Thr 172), anti-ACCα, anti-phospho-ACCα, anti-p70S6, and anti-phospho-p70S6 antibodies were purchased from Cell Signaling Technology . Monoclonal anti-HA was from Roche Molecular Biochemicals . The anti-myc antibody and the polyclonal anti-HA antibodies were from Santa Cruz Biotechnology . The anti-human synphilin-1 polyclonal antibody was generated using the human synphilin-1 fragment 34–500 aa) as the antigen as previously described 4–500 aa  was fromThe myc- or HA-tagged human full-length cDNA are in the pRK5 vector under CMV promoter as described previously ,13. HumaCells were harvested in lysis buffer  with protease inhibitors (10 µg/mL aprotinin/5 mM PMSF/10 µg/mL pepstatin/10 µg/mL leupeptin) as described previously . The resCells were harvested in a lysis buffer . Lysates were subjected to protein assays using a Bradford protein assay kit  according to the manufacturer’s protocol. An aliquot of 10 µg of protein from each sample was subjected to ATP luminescent assays using an ATP Determination Kit (Invitrogen) according to the manufacturer’s protocol. The experiments were repeated three times in duplicate.p value < 0.05 was considered significant.Quantitative data were represented as means ±Standard Error of the Mean (SEM). Protein and ATP levels were used for one-way or two-way analysis of variance (ANOVA) by Sigmastat 3.1 statistical software . Post-ANOVA analysis of group differences was performed with the Tukey test. A


Many interpretations have been proposed to explain the presence of jarosite within Martian surficial sediments, including the possibility that it precipitated within paleo-ice deposits owing to englacial weathering of dust. However, until now a similar geochemical process was not observed on Earth nor in other planetary settings. We report a multi-analytical indication of jarosite formation within deep ice. Below 1000 m depth, jarosite crystals adhering on residual silica-rich particles have been identified in the Talos Dome ice core (East Antarctica) and interpreted as products of weathering involving aeolian dust and acidic atmospheric aerosols. The progressive increase of ice metamorphism and re-crystallization with depth, favours the relocation and concentration of dust and the formation of acidic brines in isolated environments, allowing chemical reactions and mineral neo-formation to occur. This is the first described englacial diagenetic mechanism occurring in deep Antarctic ice and supports the ice-weathering model for jarosite formation on Mars, highlighting the geologic importance of paleo ice-related processes on this planet. Additional implications concern the preservation of dust-related signals in deep ice cores with respect to paleoclimatic reconstructions and the englacial history of meteorites from Antarctic blue ice fields. The authors report in-situ formation of jarosite witin the Talos Dome ice core (East Antarctica) and show that this ferric-potassium sulfate mineral is present in ice deeper than 1000 meters and progressively increases with depth. This has implications for the presence and formation mechanisms of jarosite observed on Mars. In 2004 the Opportunity rover reported widespread jarosite at Meridiani Planum2, confirming Burns’ prediction, but the geological context where jarosite was found – in fine-grained sediments within layered formations – was difficult to interpret. Since then, the mineral has been repeatedly identified on Mars5 and has been regarded as evidence for the occurrence of liquid water6 because on Earth jarosite forms as the result of low-temperature acidic-oxidative weathering of iron-bearing minerals in water-limited settings7.Jarosite, a ferric-potassium hydroxide sulfate [KFe9. The occurrence of jarosite on Mars has thus been interpreted as an indication that weathering fluids have been active over local scales and for a geologically short time interval10. An apparent paradox concerns the lithology of the protolith in which jarosite occurs, which is composed of mafic  volcanic material12. The interaction between basalt and acidic solutions has a neutralizing effect incompatible with jarosite formation13. The paradox is solved if the interaction occurs in environments isolated from bedrock, where water–rock interaction is limited and low pH is maintained during the diagenesis14.Limited water is not only necessary for the formation of jarosite, but it is critical to its long-term preservation. Experimental evidence shows that when the local water to rock ratio exceeds 10, jarosite is transformed into goethite11 or volcanic settings, such as fumaroles12. An alternative proposal predicted that jarosite formation would occur during weathering of mafic dust or fine-grained ash trapped in massive ice, but this process had not been observed until now15. The ice-weathering model suggested that the interior of ice deposits promotes acidic weathering of dust through cryo-concentration of sulfur-rich volcanic aerosols, leading to jarosite precipitation14. Experimental evidence has since shown that the weathering rate of basalt-related minerals is elevated at cryogenic temperatures16, in accordance with the identification of jarosite as a weathering product in Antarctica17. But the englacial formation of jarosite is still a speculation. Until this work, Antarctic jarosite has been found in rock varnishes and weathering rinds formed on the surface of erratics17 and of meteorites collected at blue ice fields19, or in soils developed from sulfide-rich rocks20, but not in englacial environments.Most of the hypotheses about the formation of Martian jarosite involve the interaction between acidic fluids and weathered sediments in transient lacustrine-evaporative basins similar to Earth playasHere we report the identification of jarosite in deep Antarctic ice, which we hypothesize as authigenic, resulting from the interaction between aeolian dust and acidic species trapped in the ice. The identification of the mineral is confirmed by several evidences, gathered through the application of different and independent techniques. Our results show that weathering and englacial diagenesis is possible deep inside thick ice, potentially affecting the climatic interpretation of dust records in deep ice cores. Given the similarities between our hypothesis and the Martian ice-weathering model, our findings support that ice-mediated weathering is a viable geological mechanism for jarosite formation on Mars, with major implications for past chemical weathering processes on that planet.21 and dated up to 1439 m deep , where the estimated ice age is ~153,000 years before present (BP)23  are progressively tilted and folded, and deeper than 1528 m they disappear. In addition, below 1590 m very large ice-crystals (40–50 cm), indicative of ice metamorphism, have been observed24  has been retrieved from a peripheral ice dome of the East Antarctic Plateaud24 Fig. . Ice floMost of this work is based on the identification of jarosite in dust from the deep part of TALDICE. Highlighting and quantifying the presence of jarosite within µg-size samples is analytically challenging. Evidences have been gathered through multiple techniques, so as to obtain robust and reliable results. Jarosite minerals have been observed through Scanning Electron Microscopy coupled with Energy Dispersive X-ray spectroscopy (SEM-EDX) as micrometric concretions whose shape and composition resemble jarosite. X-ray absorption spectroscopy has allowed to reconstruct the depth profile of jarosite in TALDICE, exploiting Fe speciation and coordination and a quantitative comparison with mineral standards. Scanning Transmission Electron Microscopy (STEM) and EDX, through diffraction spacings and stoichiometry, have brought a further quantitative confirmation of jarosite presence.29. Samples from the 0–1000 m (n = 20), 1000–1300 m (n = 18) and 1300–1620 m (n = 16) depth intervals present average Fe K-edge absorption energies of 7125.5(0.5), 7126.5(0.4) and 7127.2(0.3) eV (standard deviation), respectively. Considering that the positive shift between Fe2+ and Fe3+ is 4 eV, the one observed along TALDICE corresponds to a 30–40% increase of Fe3+29, in agreement with the absolute determination of Fe oxidation in TALDICE dust. Between 0 and 1000 m, Fe in mineral dust consists in a 70–30% mix of Fe3+–Fe2+, while below 1500 m only Fe3+ is detected30. The trend is indicative of in situ oxidative weathering and involves the core throughout its length, regardless of the climate-related oscillations observed in the stable isotope and dust concentration records , but also silicates  spectra 2(OH)6, as supported by EDX quantitative results (see the Supplementary Information).The morphology and elemental composition of mineral dust from sections of TALDICE deeper than 1500 m has been investigated through SEM-EDX Fig. . Weather34. Climatic proxies, such as δ18O and dust concentration, are correlated in ice cores and show coherent oscillations during glacial/interglacial cycles35. This is confirmed by TALDICE data, where their linear R-squared is 0.75 when considering the depth interval 0–1400 m  and lack of climate-related fluctuations. They are correlated with the increasing size of ice crystals along the core , which is primarily driven by ice metamorphism and not by climate36. The possibility that jarosite observed in the deep part of TALDICE comes from the bedrock is discarded, since there are no other evidences about the presence of bedrock inclusions. Only an extreme ice-folding could explain the presence of bedrock inclusions hundreds of meters above the ice-bedrock interface, but this is incompatible with the preservation of ice stratigraphy up to 1560 m deep21.Physico-chemical signals are preserved for hundreds of thousands of years within ice7. The deep part (>1000 m) of TALDICE can meet such requirements. Deep ice is affected by the progressive re-crystallization of ice grains38. A consequence of re-crystallization is that impurities incompatible with the ice lattice accumulate at ice grain junctions40 or within intra-grain µ-inclusions41. Atmospheric sulfates and sulfuric acid are strongly affected by remobilization in ice42. They are easily concentrated in isolated environments forming solutions whose eutectic temperature is below the pressure melting point of deep ice, allowing for the presence of acidic liquid layers at grain boundaries in the form of localized brines43. In deep ice, as a result of the increased temperature and ice metamorphism, the concentration and mobilization of impurities allows for the mixing of soluble and insoluble species, and for their interaction through englacial acid–base reactions45. Such small-scale environments promote the local increase of acidity and the chemical weathering of aeolian dust41. Dust particles deposited at Talos Dome have a basaltic/doleritic signature and are rich in Fe27, making deep TALDICE an environment suitable for jarosite precipitation. An additional source of acidity to the deep englacial environment could come from the oxidation of the pyrite present in TALDICE dust. Pyrite is a relatively common secondary mineral in the Victoria Land region47 and has been recognized in shallow TALDICE dust samples as an accessory mineral (see the Supplementary Information). The degradation of pyrite through the oxidation of Fe2+ to Fe3+ and the production of sulfuric acid is a common weathering reaction in glacial systems48 and could contribute to the local increase of acidity in the englacial environment. The positive shift of the Fe K-edge absorption recognized along the core , but jarosite formation and preservation requires a pH lower than 451. The only process which can explain how in deep ice such acid conditions are found, at least locally, is the concentration of acids mediated by ice re-crystallization and possibly by the oxidation of the pyrite fraction originally present in the dust deposited at Talos Dome48. Another important point concerns the occurrence of liquid water in the form of brines. Only a few direct lines of evidence for its presence in ice cores are available42 although questioned41, and theoretical supports exist43. The discrepancy could arise because while acidic brines are stable at pressure and temperature conditions found in deep ice sheets, measurements on ice cores are carried under different conditions, in particular for pressure. Yet, the occurrence of jarosite, whose formation requires liquid water, is a strong evidence for the actual, but localized, occurrence of acidic brines in deep ice, likely in correspondence of ice grain junctions or at intra-grain micro-inclusions. Our data show that once jarosite has formed, it is relatively stable in the englacial environment – at least considering the depth interval covered by TALDICE – since its contribution increases from 1000 to 1400 m deep  and then remains almost stable . Increasing temperatures can accelerate both jarosite formation and dissolution51; however, over the full range of temperatures observed within the ice sheet, jarosite is considered stable51. Instead, the factor which enables the formation of jarosite below the depth of 1000 m, is likely the presence of liquid acidic brines and the concentration of impurities in the same isolated environment, where the pressure melting point is locally lowered44. This process is linked to ice metamorphism that is known to drive the migration and the concentration of impurities42. Once mineral particles and highly concentrated acidic fluids interact, chemical weathering of minerals is enhanced, allowing for the partial dissolution of original dust particles and the precipitation of new mineral phases45, such as jarosite. Reactions involving dust in deep ice should be investigated further in relation to the preservation of climatic signals in deep ice cores, in particular considering the “quest for the oldest ice”. Ice core records concerning the analysis of Fe concentration and speciation52 could be significantly affected by the processes described here.Jarosite is present in TALDICE deeper than 1000 m, suggesting that its formation is associated with a threshold Fig. . Since j18. The rinds have been explained assuming that weathering occurred once meteorites were exposed at the ice surface, because of the interaction with acidic atmospheric species and tiny amounts of liquid water during summer18. Meteorites from blue ice can have remained into the ice for periods up to tens of thousands of years, reaching deep portions of the Antarctic ice sheet before surfacing54. The interface between deep ice and meteorites could promote the occurrence of acidic aqueous brines because of the crystallographic misfits between ice and mineral structures. Given the compositional similarity between the rinds and weathered dust in deep TALDICE, we suggest that englacial weathering should also be considered in relation to the chemical weathering of Antarctic meteorites, not only the surficial one. A support to this hypothesis comes from the Dome Fuji ice core, where jarosite has been found in correspondence of two extraterrestrial dust horizons, one interpreted as the result of a meteorite impact over East Antarctica, the other of the atmospheric entry of a fragment of an asteroid or a comet55. In TALDICE jarosite only forms below 1000 m deep; its presence in Antarctic meteorite rinds could be thus interpreted as a proxy of the englacial depth reached by meteorites themselves and of their residence time into the ice, as originally proposed by Terada et al.19.Other implications of this study concern meteorites found at Antarctic blue ice fields. On the surface of such meteorites, a weathering rind 10–100 µm thick rich in jarosite and amorphous silica is commonly found, pointing to acidic alteration14. The environment inside the Talos Dome ice is isolated from the Earth atmosphere and its conditions, including pressure, temperature, pH and chemistry, provides a suitable analogue for similar Martian settings. Dust deposited at Talos Dome is also similar to Martian atmospheric dust, being both mostly basaltic12. Within thick ice deposits it is likely that the environment would be similar at Talos Dome and under Mars-like conditions since both settings would contain at cryogenic temperatures basaltic dust and volcanogenic and biogenic (for Antarctic only) sulfur-rich aerosols. What set them apart is the oxidative agent responsible for Fe oxidation. In deep TALDICE, Fe is likely oxidized through the interaction between air bubbles and clathrates and mineral dust. On Mars oxygen is likely derived from aerosols such as oxy-chlorine species formed through UV photo-oxidation56. Considering this context, it is reasonable that the formation of jarosite on Mars involves the interaction between brines and mineral dust in deep ice, as observed in TALDICE. This mechanism for Martian jarosite precipitation is paradigm changing and strongly challenges assumptions that the mineral formed in playa settings11.The occurrence of jarosite in TALDICE supports the ice-weathering model for the formation of Martian jarosite within large ice-dust deposits58, and it has been argued that much of the physical layering observed on Mars surface could have been mediated by paleo ice-related processes59. The same could be for its mineralogical and geochemical features. In accordance with the ice-weathering model, Martian polar regions are rich in sulfate deposits which are created through the aeolian reworking of sublimation residues61. Despite these evidences and the Burns’ prophetic vision of Mars low-temperature, acid, water-limited alteration1, ice-mediated weathering has never been widely embraced or tested. Based on ongoing detections across the planet, jarosite is probably globally distributed across Mars in association with finely layered sediments5. Such sediments could result from the accumulation of weathered sublimation residue of past ice-rich deposits, since most of the settings where they occur are compatible with alteration pathways enabled by deep ice metamorphism and cryo-concentration of acidic fluids15. In contrast, recent studies have shown that the playa model does not adequately explain the chemical characteristics of jarosite rich deposits, their enormous size and the location of the putative playas62.Mars experienced dramatic climate swings and glaciations throughout its history© Millipore) and mixed with a clean electrolyte solution  until a final Na+ concentration of 1% m/m was reached. The passage is required so as to make samples electrically conductive. Each sample, consisting in 10 mL of decontaminated meltwater, was measured three times with a Beckman Multisizer 4 equipped with a quartz tube with a 30 µm orifice. Such an instrumental apparatus allows for the quantification of the insoluble particles with a spherical equivalent diameter between 0.6 and 18 µm, divided into 400 channels. Volume of insoluble particles was then converted into mass using a constant density of 2.5 g cm−3, following the convention suggested by Hänel63 for mineral aerosols and classically adopted by the ice core community65. Blanks were constantly evaluated during the analysis, their signal, on average, corresponded to 2.5% of the mean sample one. Standard deviation of the replicates ranged between 5 and 10% with respect to the total dust mass of samples. Data concerning the highest part of TALDICE  have already been published66, data from the lowest part (from 900 m to 1616 m) are published here for the first time.Dust concentration and grain size in TALDICE have been determined through Coulter counter technique in the ISO6 clean room available at the EUROCOLD facility of the University Milano-Bicocca. Twenty-five cm-long ice sections were decontaminated with three successive baths in MilliQ water  but are poorly described by log-normal distributions because of the presence of tails of coarse particles and the scarcity of finer ones (<2 µm). Examples are reported in the Supplementary Fig. 67 was applied to highlight such differences. Samples were firstly classified into upper  and lower , the algorithm was therefore instructed to reproduce the classification using the descriptive granulometric variables calculated from Coulter counter data, without considering the depth of samples. The dust grain size index shown in Fig. To highlight the granulometric differences between dust samples from the upper and lower sections of TALDICE, a dedicated index was developed. For each dust sample analyzed with Coulter counter, sixteen granulometric variables were defined, consisting in dust concentrations and ratios referred to specific grain size intervals (Supplementary Information). Surficial samples are typically fitted by a log-normal distribution, with modal values ranging between 1.8 and 2.2 µm, and present a limited tail of coarser particles (5–10 µm) related to the influence of local Antarctic dust sources68. Analyses were carried out on samples prepared extracting mineral particles from decontaminated meltwater obtained from TALDICE ice sections in an ISO6 clean room. The extraction took place with clean polycarbonate membranes  where meltwater was passed through with micro-pipettes, so as to concentrate the particles in the smallest possible area and maximize the performances69. For each sample a variable volume of meltwater was filtered depending on dust concentration , so as to have on each filter not less than 2–3 µg of dust. Membranes were then placed in clean PTFE filter-holder and mounted on the beamline. Fifty-four samples were prepared, covering the entire length of the ice core; details, including spectral data, are reported in the Supplementary Information. To avoid contamination and interference issues, the following procedures were adopted: 1- the experimental chamber was provided with a clean glove-box filled with pure nitrogen in order to handle and mount the samples in clean conditions; 2- the inner walls of the chamber were coated with clean plastic sheets to reduce the inelastic scattering produced by the interaction of the beam with the metallic chamber; 3- measurements were carried out under high vacuum conditions to remove atmospheric interferences. XAS spectra were acquired at the Fe K-edge absorption line in fluorescence mode, considering the energy interval between 7000 and 7500 eV and energy steps of 0.3 eV. The incident beam was defocused as much as possible to maximize the irradiation area, its size was 0.4 × 0.4 mm. A Vortex 4-elements silicon detector was used . The energetic calibration was monitored through the simultaneous acquisition of the absorption edge spectrum of a metallic Fe foil. For each sample–foil couple, the first derivative peak of the Fe K-edge of the foil was centered at 7112.0 eV, corrections of up to 0.2 eV were necessary. At least three spectra were acquired for each sample during different sessions and average spectra were calculated. Blank filters were measured but no Fe signal was detected. Standards  were prepared following the same procedures adopted for the samples to evaluate the precision of the method, which is 0.2 eV. Spectra were analyzed with the software Athena70. To make them comparable, fluorescence signals were processed to obtain absorption coefficients, which were normalized considering the post-edge baseline71. The pre-edge region was normalized subtracting the pre-edge baseline. The energy position of the Fe K-edge absorption was defined as the energy of the main absorption edge at the height of 0.8  with respect to the post-edge baseline. Errors shown in Fig. 72.Fe K-edge absorption energy was determined through XAS, performed at B18 beamline of the Diamond Light Source73. Specimens of the following minerals were retrieved from the collection of the University Milano-Bicocca: biotite, chlorite, glaucophane, goethite, hematite, hornblende, jarosite, limonite, magnetite, muscovite, fayalite, pargasite, pyrite, schoerlite, siderite. Fragments of the minerals were powdered using a mortar and mixed with cellulose in pellets which were measured at B18 in transmission mode. Fe K-edge absorption spectra were processed following the steps adopted for samples. To determine the amount of jarosite within the Fe minerals present in TALDICE dust, a linear combination fitting approach was followed73: spectra of the mineral standards were used to reproduce the spectra of the samples through the calculation of linear combinations with the combinatoric package of the Athena software70, based on ordinary least square regression (OLS). For each sample spectrum, all the possible combinations of 4 standards were calculated, forcing the presence of goethite, being the most common Fe-oxide within aerosols31, in particular in cold environments74. The contribution of jarosite with respect to total Fe minerals in TALDICE . Insoluble mineral dust was extracted from meltwater by filtration in an ISO6 clean room with polycarbonate membranes . After filtration some membranes were coated with gold for morphological observations (SEM) and others with graphite, for elemental mapping of mineral particles (SEM-EDX).Two samples were prepared for STEM-EDX analyses following the same procedure adopted for SEM-EDX. Dust particles from two deep sections of TALDICE (below 1500 m) were deposited on polycarbonate membranes through filtration and successively picked, embedded in low viscosity epoxy, and thin sections were placed on amorphous carbon support films and were analyzed using a JEOL 2500SE 200 kV field-emission scanning and transmission electron microscope equipped with a JEOL silicon drift detector for EDX. Diffraction data from multiple orientations were obtained from selected areas of the grains to confirm their identity. Quantitative EDX data were obtained from the same grains as the diffraction data and were quantified using standard thin-film analysis techniques with k-factors from mineral standards Table .Supplementary Information


Proximal femoral nail (PFN) is a commonly used implant for intertrochanteric fractures which is designed according to western femoral measurements. However, anthropometry of proximal femur in Indian and in general, Asian, are smaller. So a modified short PFN with smaller dimensions was developed. This study analyses the radiological and functional outcome of treatment of intertrochanteric fractures with modified short PFN.A retrospective study analysed 120 adult patients operated between 2014-2017 using modified short PFN for intertrochanteric fractures, having a minimum follow-up of 12 months. Clinical and radiological parameters including tip-apex distance (TAD), position of tip of lag screw in femoral head, lateral slide of lag screw as well as length of anti-rotation screw were measured. Final functional outcome was assessed using Barthel’s index and Kyle’s criteria.Good reduction was achieved in 90.83% cases and 79.16% had ideal placement of lag screw in femoral head. Intra-operative difficulties were encountered in 13.33% (n=16). Mean TAD AP (anteroposterior) was 11.8mm, TAD LAT  was 11.0mm and mean TAD TOT was 22.8mm. Overall mean lateral slide was 3.20mm and it was more in unstable fracture. We had five mechanical failures, one patient with screw breakage without loss of reduction and two peri-implant fractures after union. 81.66% returned to pre-injury levels of activity with 88.33% good to excellent outcome as per Kyle’s criteria.Although, not devoid of complications, modified short PFN results in good functional recovery of patients with intertrochanteric fractures of femur. Conservative management of these fractures are fraught with complications of prolonged recumbency as well as limp and shortening due to malunion in coxa-vara2. Extramedullary implants such as DHS, once considered the solution to these fractures have performed less than satisfactorily in unstable patterns paving way for intramedullary implants5. One such intramedullary implant currently in vogue is proximal femoral nail (PFN). Proximal femoral nail (Synthes) was designed keeping in mind the western population and comes in size of 240mm length, 17mm proximal diameter, distal diameter of 10-12mm and sizes of proximal and distal cephalic screws being 11mm and 6.5mm respectively. Study by Su et al demonstrated marked variability in location of femoral isthmus across various ethnic groups6. Siwach , in his study on 150 femoral bones, demonstrated smaller measurements related to proximal femoral and isthmus in Indian femurs. He recommended, in order to reduce incidence of intra-operative complications like fractures and splintering, implants need modifications according to Indian anthropometry. He recommended cephalomedullary nail to be adapted to dimensions described by Leung et al7. Leung et al used modified gamma nail for use in east Asian population and demonstrated improved clinical results in their multicentric study8. Indian femurs are proven to have considerably smaller anthropometric measurements compared to western population, thus requiring a smaller implant9. Pathrot and colleagues advised certain modifications in the short proximal femoral nail available in the Indian market10. Modified short proximal femoral nail also called trochanteric fixation nail (TFN) was introduced by Yogeshwar implants private limited for the purpose of Indian population and it works on the principles of PFN. Very few studies are published using this implant although it is being used very frequently12.Intertrochanteric fracture (IT) of femur is a very commonly encountered orthopaedic condition especially in geriatric populationWe undertook this study to assess the clinical and radiological outcomes of intertrochanteric fractures of femur treated with modified short PFN.A retrospective study was conducted in patients that were operated at our institutes with modified short proximal femoral nail for IT fractures of femur between January 2014 to December 2017. Inclusion criteria for the study was kept as skeletally mature patient, with fresh (< 2 weeks) intertrochanteric fractures, treated operatively using modified short PFN, having a minimal follow-up of at least 12 months. Exclusion criteria were, fractures extending well below lesser trochanter, associated with other fractures and inadequate medical follow-up records or radiographs.A thorough search of records were done at the respective institutes of authors  and we found that 147 patients were operated with modified short PFN during this period. However 27 records were disqualified mainly due to inadequacies in radiographs, and lack of minimum 12 months follow-up. 120 records qualified for the study. The implant used was similar to PFN (Synthes), manufactured and distributed by Yogeshwar implants private limited (Thane), except it being smaller in size . The impImmediate post-operative radiographs were considered as baseline for subsequent implant related measurements. Tip apex distance (TAD), quality of reduction, position of tip of lag screws in head was done on immediate post-operative radiograph. Sequential follow-up radiographs were evaluated to assess union, position of screws and to calculate the lateral slide of lag screws. Magnification of the radiographs were calculated dividing true lag screw width by screw width measured on radiograph. All lengths measured were multiplied by this factor to account for magnification.et al13 and adapted to cephalomedullary nail as described by Herman et al  cases. Excellent outcome was noted in 69 and good outcome in 37 patients. Twelve patients had fair outcome while two had poor outcome.Mean pre-operative Barthel index was 98±4.501 while index at final follow-up was 91.37±13.349. Although, this is a statistically significant change , clinically significant change in Barthel index was observed in 18.33% (22 cases). Ninety eight (81.66%) patients regained pre-injury status with minimal change in Barthel score (less than 5). Barthel index is scored on 10 parameters and assesses dependency of subjects in activities of daily living. It is scored on a 20-point scale and then result is multiplied by 5 to yield a score out of 100. Minimal clinically important difference (MCID) for Barthel index has been reported as 1.85 (on the 20-point Barthel index) for stroke patientsThe overall mean lateral slide of compression screws was estimated to be 3.20mm (range 0 to 13mm), after exclusion of cases with screw failure/cut-out. Unstable fracture patterns had more slide than stable ones. A1 fractures had a mean slide of 2.30mm (0-4 mm) while A2 type had 3.42mm (0-13mm).et al23 used Arbeitsgemeinschaft für Osteosynthesefragen (AO/ASIF) PFN (240mm) in a series of 46 patients and reported intra-operative difficulties in as many as 14 cases (30.4%) with fractured greater trochanter in 4(8.6%) and difficulty in nail insertion in 2 cases (4.3%). We did not encounter any greater trochanter fracture or difficulty in insertion of nail and we attribute it to smaller dimensions of the nail. Tyllianakis et al24 in a series of 45 patients had difficulty in 14 patients (31.1%) using AO/ASIF PFN. They reported fracture shaft of femur as well as fracture greater trochanter and difficult nail insertion in one case each. We did not encounter any such issue. Schipper et al25 in his large sample of 211 had difficulty during insertion of proximal screw in a mere 1.8% cases, compared to our 5% and Tyllianakis et al24 6.6%. Tyllianakis24 and Schipper25 also reported similar rate of conversion to open reduction as ours i.e. around 6%. As evident from the et al26. These studies used standard and long PFN and not the modified PFN, the absence of fracture shaft of femur, greater trochanter and difficulty inserting the nail in our present series appear noteworthy.We searched other series for intra-operative complications and compared them in et al23 also had 2 guide wire breakages. Detection of guide wire bending early is important so that it can be removed before it actually breaks. In eight cases, we were able to retrieve the wire before breaking. In such cases, free reaming beyond the bend under image intensifier may be needed after removal of guide wire. Reinsertion of a straight guide wire is necessary for screw insertion.While drilling over the guide wire, slight bending of guide wire can occur especially when it reaches near subchondral bone. It may lead to breakage of the guide wire as we saw in two of our cases and the intraosseous broken tips could not be removed. Fogagnolo Sometimes when the lower screw is placed in central portion on AP view, the proximal de rotation screw goes too superiorly. This situation may be compounded if native neck shaft angle is less than the angle of the implant or if varus reduction is accepted. Even after acceptable reduction, proximal fragment may be pushed into varus while inserting the nail and that may lead to such situations. Hence, constant watch over reduction is very important. The entry point of nail is tip of greater trochanter as it has a 6° of valgus in design. Inadvertent lateralisation of entry point not only pushes the fracture into varus but also creates mismatch of neck shaft angle between nail and bone. This leads to derotation screw trajectory going much cranial and sometimes even perforating the neck.et al27 reported that femoral shaft fractures were observed in 6 of the 107 patients with intertrochanteric fractures in their study. The nail used in current study did not cross the isthmus and no flexible reaming of medullary canal was needed. Only proximal reaming with cannulated hand held reamer sufficed the purpose. Lower dimensions of the nail averted this potentially disastrous complication.Under reaming and excessive force application during nail insertion may cause fracture shaft of femur at the tip of the nail. This also occurs due to mismatch of femoral bow and nail when it crosses the isthmus. This is true when standard implants are used in Asian patients. Yaozeng 30 of secondary surgeries after PFN with varying frequencies ranging from 3.3% by Domingo et al26 to 28.8% by Tyllianakis24. Banan et al28 reported 6.5% resurgery rate while Schipper et al25 and Fogagnolo et al23 reported at 18.4% and 20% respectively. Our study reports reoperation rate of 8.3% .Complete list of important complications encountered is tabulated in et al24 had one failure due to screw cut-out out of 46 fractures while Simmermacher et al29 had one in 191 patients (both studies used AO/ASIF PFN). Domingo et al26 showed cut-out in 4/295 and Alyassari et al30 in 4/76 , whereas Schipper et al25 found 11 failures in 211 patients. Boldin et al31 studied a sample size of 55 patients and found 3 ‘z effect’, 2 ‘reverse z effect’ and 2 screw cut-outs  leading to conclusion that anti-rotation screws being 10-15mm shorter than the lag screw prevented the cut-out. Multiple predictors of screw cut-out has been described. Using multivariate logistic regression analysis Escolar et al found TAD, suboptimal osteosynthesis and distal static locking as predictive factors for screw cut-out32. Kashigar et al used univariate analysis and found TAD, calcar-TAD, Parker’s ratio index and neck angle difference to be associated with screw cut-outs in cephalomedullary nails33. John et al studied and included biaxial cephalomedullary nails in addition to uniaxial nails. They concluded that a combination of high TAD, suboptimal position of implant and poor restoration of neck shaft angle may predispose to cut-out. However, achieving TAD within safe limits didn’t appear to influence screw and device migration in dual screw nails16. Another technical aspect of note is the length of anti-rotation screw. PFN being a twin screw construct, the smaller screw  serves the purpose of providing rotational stability while the lag screw serves load bearing function. When hip pin protrudes beyond lag screw, increased vertical forces induce Z-effect (aka Knife effect) forcing the proximal screw medially into the joint and distal lag screw to slide back laterally.Screw cut out incidences vary in literature. Tyllianakis total was found to be 22.8mm which was less than Fogagnolo et al23 and Uzen et al34 but more than Herman et al14  with screw cut-outs while others14 have refuted its use for PFN. Most of the authors however do concur with the fact that tip of lag screw must be as close to subchondral bone as possible. TAD represents both the position and depth of a screw in the femoral neck and head and was shown to be the most important predictive factor for the occurrence of a cut-out36. Geller et al37 reported a high incidence (44%) of cut-outs in intertrochanteric fractures that were surgically fixed with a TAD of >25mm.Calculations of mean TAD  reflecte19. Kyuzyk et al demonstrated that biomechanical stiffness is maximised when lag screw is placed inferiorly in AP view, and central placement in lateral view maximises its load to failure38. This position was observed in 79.16% (95/120). The next most commonly plotted position was central-central position and it was noted in 17 cases (14.16%).Ideal placement of lag screw in head is suggested to be Inferior-Centralet al39 compared screw cut-out cases with controls and found increased odds risk with lag screw position in cranial, anterior and posterior thirds of the screw. However the results did not reach statistical significance. They advocated placement in the central third of the femoral head. In our study, out of four cases where screw cut-out had occurred without infection, in three cases, the tip of lag screw was in central-central quadrant and one in central posterior quadrant. Helwig et al40 advocated advantages of cranial position in his study and is in contradiction to our findings. Further studies are required in biaxial systems to determine optimal position of the screws. However inferior central zone appears to be the safest and therefore maintenance of appropriate neck shaft angle and position of lag screw in inferior quadrant is very important aspect of the technique. Both are intricately connected as angle of screw placement is inherent to the design of neck and that is prefixed, hence unless correct neck shaft angle is achieved, screw insertion may prove to be very tricky. This again emphasises on achieving as near anatomical reduction as possible.Sub optimal position of screw in Cleveland quadrants may have a contributing effect in the screw cut-out. Zirngibl et al recommended that derotation screw must be at least 10-15mm shorter than the larger lag screw19. Zirngibl et al39 analysed this by drawing an imaginary line from tip of lag screw to the tip of nail and proved that anti-rotation pin protruding beyond this line had a significantly high odds ratio of 8.8 for fixation failure. They go on to suggest that, this could be the most important factor influencing the screw cut-out or cut-through rates. Analysis of relative screw lengths in femoral head revealed that in four out of five cases of screw cut-out, the anti-rotation screw was advanced either beyond the tip of lag screw or was at the same level, thus leading to increased vertical forces on the anti-rotation pin.As mentioned before, length of derotation screw has been reported as predictive factor for cut-out. Morihara 41, however, it is unclear what constitutes operative time in studies. Whether from incision to closure or from starting of attempt at closed reduction. In our study, we included the duration of closed reduction before incision as well. Mean blood loss is considerably lesser than that occurs with standard PFN43.In short, a combination of suboptimal position of lag screw in femoral head, high TAD as well as excessively long anti-rotation screw were found in cases that had fixation failures. The mean operative time found in this study was lesser than that reported by Fogagnolo (83.4 min) and Morihara (77min) who used standard PFN. Some studies do quote lesser operative timeIn authors’ opinion, in order to avoid screw cut-out and mechanical failure, effort needs to be directed at minimising TAD by inserting compression screw deep into the head up to 5mm below subchondral bone. In addition to ensuring adequate purchase in proximal fragment it also prevents inadvertently longer anti- rotation screws. Every effort must be directed towards careful placement of lag screw in ‘safe quadrant’ . Achieving appropriate anatomical reduction and not accepting even slight varus goes a long way in achieving this objective. Valgus reduction may be accepted, implant permitting, and may even be recommended in unstable fractures. In unstable fractures as union occurs, further impaction and varus occurs.The overall mean lateral slide of compression screws was estimated to be 3.20mm (range 0 to 13mm), after exclusion of cases with screw failure/cut-out. This lateral slide was found out to be more in unstable fractures when compared to stable fracture patterns. A1 fractures had a mean slide of 2.30mm (0-4mm) while A2 type had 3.42mm (0-13mm). As union progresses, proximal fragment gets impacted onto distal fragment as well as the nail, leading to lateral slide of both the cephalic screws and can be a surrogate marker of collapse of the fracture. Any restriction in this lateral slide may initiate cut-out or joint penetration by the screws.et al11 reported 90% excellent outcome while Pavelka et al44 had 92% excellent functional outcome.Despite these complications and mechanical failures, recovery to pre-injury functional status as per Barthel’s score was found in 81.66% of the cases (change less than MCID). As per Kyle’s criteria, good to excellent functional recovery was found in 88.33% (106) cases. Gadegon In limitations, inherent to the methodology of the study which involves medical records examination, we could not use femur length as our inclusion criteria as these were not consistently mentioned in all the records. Another limitation of this study is a lack of control group.Modified Short Proximal Femoral nail needs careful preoperative plan, followed by expert intra-operative technique coupled with good reduction. If appropriately followed, it leads to high union rate with minimal soft tissue damage. Placement of screws needs special mention and are essential for successful outcome. Safe position of screw is inferior in AP plane and central in lateral view. TAD needs to be kept to minimum. Deep insertion of lag screw into femoral head, closer to subchondral bone with a shorter anti-rotation screw which doesn’t cross the tip of lag screw is equally important. Although, not devoid of complications, modified short PFN results in good functional recovery of patients with intertrochanteric fractures of femur. The shorter nail allows for easier insertion (no reaming required post isthmus) and lesser blood loss with lesser complication rates. Its shorter length renders it not suitable for fractures that extend far distal to lesser trochanter. Further studies are needed to compare the efficacy of shorter variant in Asians as well as compare it with new variant PFNA.