When a Lump is More Than Just a Lump: Anaplastic Large Cell Lymphoma Presenting as a Pediatric Breast Mass.

Anaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin T-cell lymphoma characterized by a cluster of differentiation-30 positivity. Subtypes are characterized by positive or negative anaplastic lymphoma kinase (ALK) expression. ALCLs account for about 10% to 15% of all pediatric non-Hodgkin lymphomas and more than 90% of the cases are ALK-positive. We report a rare case of pediatric systemic ALK-negative ALCL with an atypical presentation as a painful breast mass. Despite the general benign features of most pediatric breast masses, it is important to consider malignant systemic diagnoses like the one reported here.

In vitro and in vivo evaluation of a novel wired transmission magnetically controlled capsule endoscopy system for upper gastrointestinal examination.

BACKGROUND: Magnetically controlled capsule endoscopy (MCCE) has recently increasingly been used for gastric examination. However, the image quality and esophageal observation is suboptimal. We developed a novel wired transmission magnetically controlled capsule endoscopy (WT-MCCE) system and evaluated its feasibility through in vitro and in vivo experiments. METHODS: A plastic stomach model and a pathological upper gastrointestinal model were used to evaluate the performance of WT-MCCE in vitro experiments. Twice of examination in the two in vitro models by WT-MCCE were performed by 5 endoscopists who were experienced in performing wireless capsule endoscopy. The examination of traditional gastroscopy (Olympus, GIF-HQ290) in the pathological upper gastrointestinal model was set as the control. In vivo experiments were performed in a live canine model by 3 endoscopists, in which WT-MCCE was inserted with the assistance of gastroscopy. Measurements included maneuverability, examination time, visualization of gastric mucosa, image quality and diagnostic accuracy. RESULTS: WT-MCCE showed good performance in both in vitro and in vivo experiments with excellent visualization of mucosa (75-100%). The mean operation time is 17.6 ± 2.7 min, 22.3 ± 1.9 min and 29.3 ± 3.4 min in three models, respectively. In pathological upper gastrointestinal model, all lesions, including esophageal varices, one polyp, one foreign body, two gastric ulcers and one duodenal ulcer, were detected by both WT-MCCE and traditional gastroscopy by all endoscopists. For the observation of esophagus and stomach in the canine model, WT-MCCE also showed excellent maneuverability and good image quality. CONCLUSIONS: The novel WT-MCCE system performed well in evaluating upper gastrointestinal landmarks and lesions in two in vitro models, and showed good performance in a canine model. WT-MCCE may be potentially useful for diagnosis of esophageal and gastric diseases.

Optimization of coating uniformity in a simple rotation system by using a rotating shadow mask.

A theoretically designed rotating shadow mask is proposed to optimize the uniformity of a simple rotation system, which makes full use of the width of the coating chamber. This method can fabricate a large-aperture optical component, the diameter of which is more than half the width of the coating machine. The rotating shadow mask is applied to correct the film thickness uniformity near the center point of simple plane substrate. The factors influencing the effect of the rotating shadow mask are simulated and discussed. Then the shape of the rotating shadow mask is theoretically designed, and the uniformity within a corresponding radius is well corrected. After determining the shape of the rotating shadow mask, an additional fixed shadow mask is calculated and used to improve the uniformity of the entire substrate. Through the application of the two shadow masks together, uniformity about 99.5% is obtained in the diameter of 640 mm on a 1100 mm coating machine.

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae.

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.

Characterization of microvascular disease in pediatric sickle cell disease using nailfold capillaroscopy.

Sickle cell disease (SCD) is a disorder with repetitive vaso-occlusive crises resulting in microvascular obstruction and tissue ischemia that may lead to multi-organ ischemia and dysfunction. Nailfold videocapillaroscopy (NFC) is an imaging technique utilized in clinical rheumatology to visualize capillaries located near the fingertip. To characterize NFC abnormalities in the setting of pediatric SCD, we performed NFC using a video capillaroscope on 8 digits in 44 stable SCD patients and 65 age matched healthy controls. Mean capillary number was lower (6.4 ± 1.3 vs 7.5 ± 1.8, p = 0.001) in the SCD group compared to controls. The percentage of dilated capillaries was similar (7.1 ± 8.3 vs. 5.9 ± 8.2, p = 0.4). The large majority of capillaries visualized in the SCD and control groups were normal capillary types per the EULAR definition, with a similar percentage of normal, nonspecific capillary morphologies and abnormal types. Regarding normal capillary sub-types, the SCD group and controls exhibited similar percentages of stereotype hairpin shapes, and tortuous or once or twice crossing type capillaries. On multivariate analyses, mean capillary number was independently associated with SCD after adjusting for age, body mass index, systolic blood pressure and gender. In conclusion, pediatric SCD is associated with lower capillary number but similar percentage of dilated capillaries and morphology on NFC. In our SCD cohort, capillary number was unrelated to our available markers of disease severity, including history of sickle crises, previous hospitalization for crises or Hemoglobin F levels.

Regulated redirection of central carbon flux enhances anaerobic production of bioproducts in Zymomonas mobilis.

The microbial production of chemicals and fuels from plant biomass offers a sustainable alternative to fossilized carbon but requires high rates and yields of bioproduct synthesis. Z. mobilis is a promising chassis microbe due to its high glycolytic rate in anaerobic conditions that are favorable for large-scale production. However, diverting flux from its robust ethanol fermentation pathway to nonnative pathways remains a major engineering hurdle. To enable controlled, high-yield synthesis of bioproducts, we implemented a central-carbon metabolism control-valve strategy using regulated, ectopic expression of pyruvate decarboxylase (Pdc) and deletion of chromosomal pdc. Metabolomic and genetic analyses revealed that glycolytic intermediates and NADH accumulate when Pdc is depleted and that Pdc is essential for anaerobic growth of Z. mobilis. Aerobically, all flux can be redirected to a 2,3-butanediol pathway for which respiration maintains redox balance. Anaerobically, flux can be redirected to redox-balanced lactate or isobutanol pathways with ≥65% overall yield from glucose. This strategy provides a promising path for future metabolic engineering of Z. mobilis.

OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate.

Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

Massive Splenic Infarction in a Child With Sickle Cell Disease on Chronic Transfusion Therapy.

Massive splenic infarction (MSI) is a rare complication of sickle cell disease, as the spleen generally atrophies within the first few years of life. We report a case of MSI in a 12-year-old boy with homozygous sickle cell anemia (Hb SS) whose chronic transfusion therapy resulted in hypersplenism. The occurrence of a complicated MSI in our patient should perhaps further encourage elective splenectomy in such patients, despite known potential perioperative complications and postsplenectomy risks of infection and thrombosis.

Correction: Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

[This corrects the article DOI: 10.1371/journal.pgen.1006372.].

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Advances and prospects in metabolic engineering of Zymomonas mobilis.

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of "Design-Build-Test-Learn", as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era.

Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

BACKGROUND: Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. RESULTS: Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. CONCLUSIONS: We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain. This strain represents a xylose fermenting yeast specifically tailored to GVL produced hydrolysates.

Leveraging Genetic-Background Effects in Saccharomyces cerevisiae To Improve Lignocellulosic Hydrolysate Tolerance.

UNLABELLED: A major obstacle to sustainable lignocellulosic biofuel production is microbe inhibition by the combinatorial stresses in pretreated plant hydrolysate. Chemical biomass pretreatment releases a suite of toxins that interact with other stressors, including high osmolarity and temperature, which together can have poorly understood synergistic effects on cells. Improving tolerance in industrial strains has been hindered, in part because the mechanisms of tolerance reported in the literature often fail to recapitulate in other strain backgrounds. Here, we explored and then exploited variations in stress tolerance, toxin-induced transcriptomic responses, and fitness effects of gene overexpression in different Saccharomyces cerevisiae (yeast) strains to identify genes and processes linked to tolerance of hydrolysate stressors. Using six different S. cerevisiae strains that together maximized phenotypic and genetic diversity, first we explored transcriptomic differences between resistant and sensitive strains to identify common and strain-specific responses. This comparative analysis implicated primary cellular targets of hydrolysate toxins, secondary effects of defective defense strategies, and mechanisms of tolerance. Dissecting the responses to individual hydrolysate components across strains pointed to synergistic interactions between osmolarity, pH, hydrolysate toxins, and nutrient composition. By characterizing the effects of high-copy gene overexpression in three different strains, we revealed the breadth of the background-specific effects of gene fitness contributions in synthetic hydrolysate. Our approach identified new genes for engineering improved stress tolerance in diverse strains while illuminating the effects of genetic background on molecular mechanisms. IMPORTANCE: Recent studies on natural variation within Saccharomyces cerevisiae have uncovered substantial phenotypic diversity. Here, we took advantage of this diversity, using it as a tool to infer the effects of combinatorial stress found in lignocellulosic hydrolysate. By comparing sensitive and tolerant strains, we implicated primary cellular targets of hydrolysate toxins and elucidated the physiological states of cells when exposed to this stress. We also explored the strain-specific effects of gene overexpression to further identify strain-specific responses to hydrolysate stresses and to identify genes that improve hydrolysate tolerance independent of strain background. This study underscores the importance of studying multiple strains to understand the effects of hydrolysate stress and provides a method to find genes that improve tolerance across strain backgrounds.

Global transcriptome analysis and enhancer landscape of human primary T follicular helper and T effector lymphocytes.

T follicular helper (Tfh) cells are a subset of CD4(+) T helper cells that migrate into germinal centers and promote B-cell maturation into memory B and plasma cells. Tfh cells are necessary for promotion of protective humoral immunity following pathogen challenge, but when aberrantly regulated, drive pathogenic antibody formation in autoimmunity and undergo neoplastic transformation in angioimmunoblastic T-cell lymphoma and other primary cutaneous T-cell lymphomas. Limited information is available on the expression and regulation of genes in human Tfh cells. Using a fluorescence-activated cell sorting-based strategy, we obtained primary Tfh and non-Tfh T effector cells from tonsils and prepared genome-wide maps of active, intermediate, and poised enhancers determined by chromatin immunoprecipitation-sequencing, with parallel transcriptome analyses determined by RNA sequencing. Tfh cell enhancers were enriched near genes highly expressed in lymphoid cells or involved in lymphoid cell function, with many mapping to sites previously associated with autoimmune disease in genome-wide association studies. A group of active enhancers unique to Tfh cells associated with differentially expressed genes was identified. Fragments from these regions directed expression in reporter gene assays. These data provide a significant resource for studies of T lymphocyte development and differentiation and normal and perturbed Tfh cell function.

Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by Rhodopseudomonas palustris.

Lignocellulosic biomass hydrolysates hold great potential as a feedstock for microbial biofuel production, due to their high concentration of fermentable sugars. Present at lower concentrations are a suite of aromatic compounds that can inhibit fermentation by biofuel-producing microbes. We have developed a microbial-mediated strategy for removing these aromatic compounds, using the purple nonsulfur bacterium Rhodopseudomonas palustris. When grown photoheterotrophically in an anaerobic environment, R. palustris removes most of the aromatics from ammonia fiber expansion (AFEX) treated corn stover hydrolysate (ACSH), while leaving the sugars mostly intact. We show that R. palustris can metabolize a host of aromatic substrates in ACSH that have either been previously described as unable to support growth, such as methoxylated aromatics, and those that have not yet been tested, such as aromatic amides. Removing the aromatics from ACSH with R. palustris, allowed growth of a second microbe that could not grow in the untreated ACSH. By using defined mutants, we show that most of these aromatic compounds are metabolized by the benzoyl-CoA pathway. We also show that loss of enzymes in the benzoyl-CoA pathway prevents total degradation of the aromatics in the hydrolysate, and instead allows for biological transformation of this suite of aromatics into selected aromatic compounds potentially recoverable as an additional bioproduct.

Metabolic mechanism of Cr(VI) pollution remediation by Alicycliphilus denitrificans Ylb10.

Cr(VI) is a well-known toxic pollutant and its remediation has attracted great attention. It is important to continuously discover and explore new high-efficiency Cr(VI) reducing bacteria to further improve the efficiency of Cr(VI) pollution remediation. In this paper, metabolic mechanism of Cr(VI) reduction in a new highly efficient Cr(VI) reducing bacterium, Alicycliphilus denitrificans Ylb10, was investigated. The results showed that Ylb10 could tolerate and completely reduce 450 mg/L Cr(VI). Cr(VI) can be reduced in the intracellular compartment, membrane and the extracellular compartment, with the plasma membrane being the main active site for Cr(VI) reduction. With the addition of NADH, the reduction efficiency of cell membrane components for Cr(VI) increased 2.3-fold. The omics data analysis showed that sulfite reductase CysJ, thiosulfate dehydrogenase TsdA, nitrite reductase NrfA, nitric oxide reductase NorB, and quinone oxidoreductase ChrR play important roles in the reduction of Cr(VI), in the intracellular, and the extracellular compartment, and the membrane of Ylb10, and therefore Cr(VI) was reduced by the combined action of several reductases at these three locations.

Authentication and recovery of medical diagnostic image using dual reversible digital watermarking.

This paper proposes a new region-based tampering detection and recovering method that utilizes both reversible digital watermarking and quad-tree decomposition for medical diagnostic image's authentication. Firstly, the quad-tree decomposition is used to divide the original image into blocks with high homogeneity, and then we computer pixels' linear interpolation as each block's recovery feature. Secondly, these recovery features as the first layer watermarking information is embedded by using simple invertible integer transformation. In order to enhance the proposed method's security, the logistic chaotic map is exploited to choose each block's reference pixel. The second layer watermark comprises by the quad-tree information and essential parameters for extraction are embedded by LSB replacement. In the authentication phase, the embedded watermark is extracted and the source image is recovered, and the similar linear interpolation technique is utilized to get each block's feature. Therefore, the tampering detection and localization can be achieved through comparing the extracted feature with the recomputed one, and the extracted feature can be used to recover those tampered regions with high similarity to their original state. Experimental results show that, compared with previous similar existing scheme, the proposed method not only achieves high embedding capacity and good visual quality of marked and restored image, but also has more accuracy for tampering detection.

The effects of modified Guizhi plus Gegen decoction combined with the blade needle therapy on TCM syndromes, cervical curvature and levels of inflammatory factors in patients with cervical spondylotic radiculopathy.

OBJECTIVE: To explore the effect of modified Guizhi plus Gegen decoction combined with the blade needle therapy on traditional Chinese medicine (TCM) syndromes, cervical curvature, and inflammatory factor levels in patients with cervical spondylotic radiculopathy. METHODS: In this retrospective study, 114 patients with cervical spondylotic radiculopathy who visited Pain Clinic, Hangzhou Fuyang Hospital of TCM Orthopedics and Traumatology from January 2020 to December 2022 were selected as the study subjects. According to different treatment methods, these patients were divided into an observation group (n=57, treated with blade needle therapy) and a control group (n=57, treated with modified Guizhi plus Gegen decoction combined with the blade needle therapy). Patients in both groups were treated for 3 courses. The treatment effects, TCM syndrome scores, cervical curvature, hemorheology indexes, inflammatory factors and adverse reactions were analyzed and compared between the two groups. RESULTS: The effective rate of patients in the observation group was 94.74%, which was significantly higher than 82.46% in the control group (P<0.05). After treatment, TCM syndrome scores, hemorheology indexes, and inflammatory factors levels in both of groups were significantly decreased in contrast to before treatment, while the cervical curvature was obviously increased. Compared with the control group, after the treatment, TCM syndrome scores, hemorheology indexes, inflammatory factors levels after treatment in the observation group were obviously lower, while the cervical curvature in the observation group being significantly increased (all P<0.05). No statistical differences were found for the incidence of adverse reactions between two the groups. CONCLUSION: Modified Guizhi plus Gegen decoction combined with the blade needle therapy effectively improved the TCM syndrome scores, restored the curvature of the cervical spine, improved the hemorheology of patients, inhibited the levels of inflammatory factors and it also has few adverse reactions, with a significant treatment effect in patients with cervical spondylotic radiculopathy.

Study on the Damage Model of Non-Persistent Jointed Rock Mass under the Coupling of Freeze-Thaw and Shear.

Considering that a jointed rock mass in a cold area is often affected by periodic freeze-thaw cycles and shear failure, definitions for the mesoscopic and macroscopic damage to a jointed rock mass under the coupling of freeze-thaw and shear are proposed, and the damage mechanism is verified according to experimental results. The results show that: (1) the jointed rock specimens increase macro-joints and meso-defects, the mechanical properties deteriorate significantly under freeze-thaw cycles, and the damage degree becomes more and more significant with the increases in freeze-thaw cycles and joint persistency. (2) When the number of freeze-thaw cycles is constant, the total damage variable value gradually increases with the increase in joint persistency. The damage variable difference in specimens with different persistency is distinct, which is gradually reduced in the later cycles, indicating a weakening influence of persistency on the total damage variable. (3) The shear resistance of non-persistent jointed rock mass in a cold area is determined by the coupling effect of meso-damage and frost heaving macro-damage. The coupling damage variable can accurately describe the damage variation law of jointed rock mass under freeze-thaw cycles and shear load.

The robustness of the Escherichia coli signal-transducing UTase/UR-PII covalent modification cycle to variation in the PII concentration requires very strong inhibition of the UTase activity of UTase/UR by glutamine.

Uridylyltransferase/uridylyl-removing enzyme (UTase/UR) catalyzes uridylylation of PII and deuridylylation of PII-UMP, with both activities regulated by glutamine. In a reconstituted UTase/UR-PII cycle containing wild-type UTase/UR, the steady-state modification of PII varied from nearly complete modification to nearly complete demodification as glutamine was varied, whether the level of PII was saturating or unsaturating, but when a His-tagged version of UTase/UR was used, the robustness to variations in PII concentration was lost and the range of PII modification states in response to glutamine became smaller as the PII concentration increased. The presence of the His tag on UTase/UR did not alter PII substrate inhibition of the UT activity and had little effect on the level of the UT activity but resulted in a slight defect in UR activity. Importantly, at high PII concentrations, glutamine inhibition of the UT activity was incomplete. We hypothesized that binding of PII to the UR active site in the HD domain was responsible for PII substrate inhibition of the UT activity and, in the His-tagged enzyme, also weakened glutamine inhibition of the UT activity. Consistent with this, three different UTase/UR proteins with HD domain alterations lacked substrate inhibition of UT activity by PII; in one case, the HD alteration eliminated glutamine regulation of UT activity, while for the other two proteins, alterations of the HD domain partially compensated for the effect of the His tag in restoring glutamine regulation of UT activity. We conclude that very strong inhibition of UT activity was required for the UTase/UR-PII cycle to display robustness to the PII concentration, that in the wild-type enzyme PII brings about substrate inhibition of the UT activity by binding to the HD domain of the enzyme, and that addition of an N-terminal His tag resulted in an altered enzyme with subtle changes in the interactions between domains such that binding of PII to the HD domain interfered with glutamine regulation of the UT domain.