In-situ construction of cation vacancies in amphoteric-metallic element-doped NiFe-LDH as ultrastable and efficient alkaline hydrogen evolution electrocatalysts at 1000 mA cm-2.

Developing efficient and stable electrocatalysts at affordable costs is very important for large-scale production of green hydrogen. In this study, unique amphoteric metallic element-doped NiFe-LDH nanosheet arrays (NiFeCd-LDH, NiFeZn-LDH and NiFeAl-LDH) using as high-performance bifunctional electrocatalysts for hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) were reported, by tuning electronic structure and vacancy engineering. It was found that NiFeCd-LDH possesses the lowest overpotentials of 85 mV and 240 mV (at 10 mA cm-2) for HER and OER, respectively. Density functional theory (DFT) calculations reveal the synergistic effect of Cd vacancies and Cd doping on improving alkaline HER performance, which promote the achievement of excellent catalytic activity and ultrastable hydrogen production at a large current density of 1000 mA cm-2 within 250 h. Besides, the overall water splitting performance of the as-prepared NiFeCd-LDH requires only 1.580 V to achieve a current density of 10 mA cm-2 in alkaline seawater media, underscoring the importance of modifying the electronic properties of LDH for efficient overall water splitting in both alkaline water/seawater environments.

A high-throughput cigarette smoke-treated bronchosphere model for disease-relevant phenotypic compound screening.

Cigarette smoking (CS) is the leading cause of COPD, and identifying the pathways that are driving pathogenesis in the airway due to CS exposure can aid in the discovery of novel therapies for COPD. An additional barrier to the identification of key pathways that are involved in the CS-induced pathogenesis is the difficulty in building relevant and high throughput models that can recapitulate the phenotypic and transcriptomic changes associated with CS exposure. To identify these drivers, we have developed a cigarette smoke extract (CSE)-treated bronchosphere assay in 384-well plate format that exhibits CSE-induced decreases in size and increase in luminal secretion of MUC5AC. Transcriptomic changes in CSE-treated bronchospheres resemble changes that occur in human smokers both with and without COPD compared to healthy groups, indicating that this model can capture human smoking signature. To identify new targets, we ran a small molecule compound deck screening with diversity in target mechanisms of action and identified hit compounds that attenuated CSE induced changes, either decreasing spheroid size or increasing secreted mucus. This work provides insight into the utility of this bronchopshere model to examine human respiratory disease impacted by CSE exposure and the ability to screen for therapeutics to reverse the pathogenic changes caused by CSE.

Deformation constitutive model of subgrade soil under intermittent cyclic loading.

During the actual operation of subways, high-speed railway, and other infrastructure, subgrade soil will be subjected to periodic vibration and intermittent loading, i.e., intermittent cyclic load. To study the deformation characteristics of subgrade soil under intermittent cyclic loading, the Newtonian dashpot in the generalized Kelvin model is replaced by the fractional Abel dashpot, a fractional generalized Kelvin model is established. Then, a nonlinear viscoplastic body model considering damage is proposed, which is connected in series with the improved fractional generalized Kelvin model, and the deformation constitutive model of soil under intermittent cyclic loading is obtained. Finally, the effectiveness and applicability of the model are analyzed, and the model is compared with other constitutive models. The results show that the constitutive model established in the paper can reflect the combined effect of confining pressure, frequency, dynamic stress amplitude and other factors on the cumulative plastic strain of soil, and can well characterize the development law of three types of cumulative plastic strain curves of soil: stable, critical, and failure. The fitting parameters [Formula: see text] and [Formula: see text] tend to decay and rise, respectively, with the increase in loading stage N, which reflects the rationality and accuracy of the model.

Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition.

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

MS Location Estimation Based on the Artificial Bee Colony Algorithm.

With the mature technology of wireless communications, the function of estimating the mobile station (MS) position has become essential. Suppressing the bias resulting from non-line-of-sight (NLSO) scenarios is the main issue for a wireless location network. The artificial bee colony (ABC) algorithm, based on the depiction of bee swarm's foraging characteristics, is widely applied to solve optimization problems in several fields. Based on three measurements of time-of-arrival (TOA), an objective function is used to quantify the additional NLOS error on the MS positioning scheme. The ABC algorithm is adopted to locate the most precise MS location by minimizing the objective function value. The performance of the proposed positioning methods is verified under various error distributions through computer simulations. Meanwhile, the localization accuracy achieved by other existing methods is also investigated. According to the simulation results, accurate estimation of the MS position is derived and therefore the efficiency of the localization process is increased.

A cell-level quality control workflow for high-throughput image analysis.

BACKGROUND: Image-based high throughput (HT) screening provides a rich source of information on dynamic cellular response to external perturbations. The large quantity of data generated necessitates computer-aided quality control (QC) methodologies to flag imaging and staining artifacts. Existing image- or patch-level QC methods require separate thresholds to be simultaneously tuned for each image quality metric used, and also struggle to distinguish between artifacts and valid cellular phenotypes. As a result, extensive time and effort must be spent on per-assay QC feature thresholding, and valid images and phenotypes may be discarded while image- and cell-level artifacts go undetected. RESULTS: We present a novel cell-level QC workflow built on machine learning approaches for classifying artifacts from HT image data. First, a phenotype sampler based on unlabeled clustering collects a comprehensive subset of cellular phenotypes, requiring only the inspection of a handful of images per phenotype for validity. A set of one-class support vector machines are then trained on each biologically valid image phenotype, and used to classify individual objects in each image as valid cells or artifacts. We apply this workflow to two real-world large-scale HT image datasets and observe that the ratio of artifact to total object area (ARcell) provides a single robust assessment of image quality regardless of the underlying causes of quality issues. Gating on this single intuitive metric, partially contaminated images can be salvaged and highly contaminated images can be excluded before image-level phenotype summary, enabling a more reliable characterization of cellular response dynamics. CONCLUSIONS: Our cell-level QC workflow enables identification of artificial cells created not only by staining or imaging artifacts but also by the limitations of image segmentation algorithms. The single readout ARcell that summaries the ratio of artifacts contained in each image can be used to reliably rank images by quality and more accurately determine QC cutoff thresholds. Machine learning-based cellular phenotype clustering and sampling reduces the amount of manual work required for training example collection. Our QC workflow automatically handles assay-specific phenotypic variations and generalizes to different HT image assays.

Open-source discovery of chemical leads for next-generation chemoprotective antimalarials.

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification.

The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts. The new pipeline was first applied to ~100 large-scale historical HTS assays; in silico analysis showed auto-QC led to a noticeably stronger structure-activity relationship. The method was further tested in several independent HTS runs, where QC results were sampled for experimental validation. Significantly increased hit confirmation rates were obtained after the QC steps, confirming that the proposed method was effective in enriching true-positive hits. An implementation of the algorithm is available to the screening community.

All-optical 1st- and 2nd-order differential equation solvers with large tuning ranges using Fabry-Pérot semiconductor optical amplifiers.

We experimentally demonstrate an all-optical temporal computation scheme for solving 1st- and 2nd-order linear ordinary differential equations (ODEs) with tunable constant coefficients by using Fabry-Pérot semiconductor optical amplifiers (FP-SOAs). By changing the injection currents of FP-SOAs, the constant coefficients of the differential equations are practically tuned. A quite large constant coefficient tunable range from 0.0026/ps to 0.085/ps is achieved for the 1st-order differential equation. Moreover, the constant coefficient p of the 2nd-order ODE solver can be continuously tuned from 0.0216/ps to 0.158/ps, correspondingly with the constant coefficient q varying from 0.0000494/ps(2) to 0.006205/ps(2). Additionally, a theoretical model that combining the carrier density rate equation of the semiconductor optical amplifier (SOA) with the transfer function of the Fabry-Pérot (FP) cavity is exploited to analyze the solving processes. For both 1st- and 2nd-order solvers, excellent agreements between the numerical simulations and the experimental results are obtained. The FP-SOAs based all-optical differential-equation solvers can be easily integrated with other optical components based on InP/InGaAsP materials, such as laser, modulator, photodetector and waveguide, which can motivate the realization of the complicated optical computing on a single integrated chip.

Design of an ultra-short coupler in an asymmetric twin-waveguide structure using transformation optics.

An integrated vertical coupler that transfers light from the lower passive waveguide to the upper active waveguide in an asymmetric twin-waveguide integration structure (in InGaAsP/InP material system) is designed using transformation optics (TO). The length of the coupler is as short as 3 µm, which is two orders of magnitude shorter than that of traditional tapered couplers. According to three-dimensional full-wave simulations, the designed optimized coupler has a high coupling efficiency of 94.9%, and a low reflection at the wavelength of 1.55 µm. Subsequently, quasi-conformal mapping is employed to reduce the material complexity and to make it possible to realize the coupler by purely using an isotropic dielectric material. Applying TO to integrated photonic devices may motivate new applications, and improve integration density on the InP platform.

Gastro-floating tablets of cephalexin: preparation and in vitro/in vivo evaluation.

Gastro-floating tablets of cephalexin were developed to prolong the residence time in major absorption sites. Gastro-floating tablets were prepared and optimized using hydroxypropyl methylcellulose (HPMC K100M) as matrix and sodium bicarbonate as a gas-forming agent. The properties of the tablets in terms of floating lag time, floating time and in vitro release were evaluated. Furthermore, in vivo pharmacokinetic study in fed and fasted beagle dogs was performed. The gastro-floating tablets had short floating lag time and exhibited a satisfactory sustained-release profile in vitro. Compared with conventional capsules, the gastro-floating tablets presented a sustained-release behavior with a relative bioavailability of 99.4%, while the reference sustained-release tablets gave a relative bioavailability of only 39.3%. Meanwhile, the food had significant effect on the pharmacokinetics of sustained-release tablets. It was concluded that the gastro-floating tablets had a sustained-release effect in vitro and in vivo, as well as desired pharmacokinetic properties in both fed and fasted conditions.

Haloperidol prophylaxis decreases delirium incidence in elderly patients after noncardiac surgery: a randomized controlled trial*.

OBJECTIVES: To evaluate the efficacy and safety of short-term low-dose intravenous haloperidol for delirium prevention in critically ill elderly patients after noncardiac surgery. DESIGN: Prospective, randomized, double-blind, and placebo-controlled trial in two centers. SETTING: Intensive care units of two large tertiary teaching hospitals. PATIENTS: Four hundred fifty-seven patients 65 yrs or older who were admitted to the intensive care unit after noncardiac surgery. INTERVENTION: Haloperidol (0.5 mg intravenous bolus injection followed by continuous infusion at a rate of 0.1 mg/h for 12 hrs; n = 229) or placebo (n = 228) was randomly administered from intensive care unit admission. MEASURES: The primary end point was the incidence of delirium within the first 7 days after surgery. Secondary end points included time to onset of delirium, number of delirium-free days, length of intensive care unit stay, all-cause 28-day mortality, and adverse events. Delirium was assessed using the confusion assessment method for the intensive care unit. RESULTS: The incidence of delirium during the first 7 days after surgery was 15.3% (35/229) in the haloperidol group and 23.2% (53/228) in the control group (p = .031). The mean time to onset of delirium and the mean number of delirium-free days were significantly longer (6.2 days [95% confidence interval 5.9-6.4] vs. 5.7 days [95% confidence interval 5.4-6.0]; p = .021; and 6.8 ± 0.5 days vs. 6.7 ± 0.8 days; p = .027, respectively), whereas the median length of intensive care unit stay was significantly shorter (21.3 hrs [95% confidence interval 20.3-22.2] vs. 23.0 hrs [95% confidence interval 20.9-25.1]; p = .024) in the haloperidol group than in the control group. There was no significant difference with regard to all-cause 28-day mortality between the two groups (0.9% [2/229] vs. 2.6% [6/228]; p = .175). No drug-related side effects were documented. CONCLUSIONS: For elderly patients admitted to intensive care unit after noncardiac surgery, short-term prophylactic administration of low-dose intravenous haloperidol significantly decreased the incidence of postoperative delirium. The therapy was well-tolerated.

Incidence and risk factors of delirium in critically ill patients after non-cardiac surgery.

BACKGROUND: Delirium is a common and deleterious complication in critically ill patients after surgery. The purpose of this study was to determine the incidence and risk factors of delirium in critically ill patients after non-cardiac surgery, and to investigate the relationship between the serum cortisol level and the occurrence of postoperative delirium. METHODS: In a prospective cohort study, 164 consecutive patients who were admitted to the surgical intensive care unit after non-cardiac surgery were enrolled. Baseline characteristics and perioperative variables were collected. Blood samples were obtained on the first postoperative day and serum cortisol concentrations were measured. Delirium was assessed using the Nursing Delirium Screening Scale until the seventh postoperative day or the disappearance of delirious symptoms. RESULTS: Postoperative delirium occurred in 44.5% of patients (73 of 164). The median time to first onset of delirium is 0 (range 0 to 5 days) and the median duration of delirium is 3 (1 to 13) days. Independent risk factors of postoperative delirium included increasing age (odds ratio (OR) 2.646, 95% confidence interval (CI) 1.431 to 4.890, P = 0.002), a history of previous stroke (OR 4.499, 95%CI 1.228 to 16.481, P = 0.023), high Acute Physiology and Chronic Health Evaluation II score on surgical intensive care unite admission (OR 1.391, 95%CI 1.201 to 1.612, P < 0.001), and high serum cortisol level on the 1st postoperative day (OR 3.381, 95%CI 1.690 to 6.765, P = 0.001). The development of delirium was linked to higher incidence of postoperative complications (28.8% vs. 7.7%, P < 0.001), and longer duration of hospitalization (18 (7 to 74) days vs. 13 (3 to 48) days, P < 0.001). CONCLUSIONS: Delirium was a frequent complication in critically ill patients after non-cardiac surgery. High serum cortisol level was associated with increased incidence of postoperative delirium.

Evidence-based annotation of the malaria parasite's genome using comparative expression profiling.

A fundamental problem in systems biology and whole genome sequence analysis is how to infer functions for the many uncharacterized proteins that are identified, whether they are conserved across organisms of different phyla or are phylum-specific. This problem is especially acute in pathogens, such as malaria parasites, where genetic and biochemical investigations are likely to be more difficult. Here we perform comparative expression analysis on Plasmodium parasite life cycle data derived from P. falciparum blood, sporozoite, zygote and ookinete stages, and P. yoelii mosquito oocyst and salivary gland sporozoites, blood and liver stages and show that type II fatty acid biosynthesis genes are upregulated in liver and insect stages relative to asexual blood stages. We also show that some universally uncharacterized genes with orthologs in Plasmodium species, Saccharomyces cerevisiae and humans show coordinated transcription patterns in large collections of human and yeast expression data and that the function of the uncharacterized genes can sometimes be predicted based on the expression patterns across these diverse organisms. We also use a comprehensive and unbiased literature mining method to predict which uncharacterized parasite-specific genes are likely to have roles in processes such as gliding motility, host-cell interactions, sporozoite stage, or rhoptry function. These analyses, together with protein-protein interaction data, provide probabilistic models that predict the function of 926 uncharacterized malaria genes and also suggest that malaria parasites may provide a simple model system for the study of some human processes. These data also provide a foundation for further studies of transcriptional regulation in malaria parasites.

In silico discovery of transcription regulatory elements in Plasmodium falciparum.

BACKGROUND: With the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (approximately 90% AT) presents significant challenges to in silico cis-regulatory element discovery. RESULTS: We have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted. CONCLUSION: This GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that cis-regulatory elements play a role in the transcriptional control of many P. falciparum biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

Large-scale annotation of small-molecule libraries using public databases.

While many large publicly accessible databases provide excellent annotation for biological macromolecules, the same is not true for small chemical compounds. Commercial data sources also fail to encompass an annotation interface for large numbers of compounds and tend to be cost prohibitive to be widely available to biomedical researchers. Therefore, using annotation information for the selection of lead compounds from a modern day high-throughput screening (HTS) campaign presently occurs only under a very limited scale. The recent rapid expansion of the NIH PubChem database provides an opportunity to link existing biological databases with compound catalogs and provides relevant information that potentially could improve the information garnered from large-scale screening efforts. Using the 2.5 million compound collection at the Genomics Institute of the Novartis Research Foundation (GNF) as a model, we determined that approximately 4% of the library contained compounds with potential annotation in such databases as PubChem and the World Drug Index (WDI) as well as related databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) and ChemIDplus. Furthermore, the exact structure match analysis showed 32% of GNF compounds can be linked to third party databases via PubChem. We also showed annotations such as MeSH (medical subject headings) terms can be applied to in-house HTS databases in identifying signature biological inhibition profiles of interest as well as expediting the assay validation process. The automated annotation of thousands of screening hits in batch is becoming feasible and has the potential to play an essential role in the hit-to-lead decision making process.

Investigation of second-order nonlinearity in poled-polymer during photobleaching.

The second-order nonlinearity of poled-polymer during photobleaching process is monitored with second harmonic generation(SHG) intensity enhanced by surface plasmon resonance(SPR) in an attenuated total reflection (ATR) configuration. The nonlinear coefficient d(33) of the poled polymer is determined by comparing the SPR enhanced second harmonic generation(SHG) intensities with that from the calibrated quartz. Experimental results show that photobleaching process has less effect on the second-order nonlinearity of a poled cross-linked polymer than that of a poled side-chain polymer.

In silico gene function prediction using ontology-based pattern identification.

MOTIVATION: With the emergence of genome-wide expression profiling data sets, the guilt by association (GBA) principle has been a cornerstone for deriving gene functional interpretations in silico. Given the limited success of traditional methods for producing clusters of genes with great amounts of functional similarity, new data-mining algorithms are required to fully exploit the potential of high-throughput genomic approaches. RESULTS: Ontology-based pattern identification (OPI) is a novel data-mining algorithm that systematically identifies expression patterns that best represent existing knowledge of gene function. Instead of relying on a universal threshold of expression similarity to define functionally related groups of genes, OPI finds the optimal analysis settings that yield gene expression patterns and gene lists that best predict gene function using the principle of GBA. We applied OPI to a publicly available gene expression data set on the life cycle of the malarial parasite Plasmodium falciparum and systematically annotated genes for 320 functional categories based on current Gene Ontology annotations. An ontology-based hierarchical tree of the 320 categories provided a systems-wide biological view of this important malarial parasite.

Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential.

Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.

Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.