Wheat yellow mosaic virus NIb targets TaVTC2 to elicit broad-spectrum pathogen resistance in wheat.

GDP-L-galactose phosphorylase (VTC2) catalyses the conversion of GDP-L-galactose to L-galactose-1-P, a vital step of ascorbic acid (AsA) biosynthesis in plants. AsA is well known for its function in the amelioration of oxidative stress caused by most pathogen infection, but its function against viral infection remains unclear. Here, we have identified a VTC2 gene in wheat named as TaVTC2 and investigated its function in association with the wheat yellow mosaic virus (WYMV) infection. Our results showed that overexpression of TaVTC2 significantly increased viral accumulation, whereas knocking down TaVTC2 inhibited the viral infection in wheat, suggesting a positive regulation on viral infection by TaVTC2. Moreover, less AsA was produced in TaVTC2 knocking down plants (TaVTC2-RNAi) which due to the reduction in TaVTC2 expression and subsequently in TaVTC2 activity, resulting in a reactive oxygen species (ROS) burst in leaves. Furthermore, the enhanced WYMV resistance in TaVTC2-RNAi plants was diminished by exogenously applied AsA. We further demonstrated that WYMV NIb directly bound to TaVTC2 and inhibited TaVTC2 enzymatic activity in vitro. The effect of TaVTC2 on ROS scavenge was suppressed by NIb in a dosage-dependent manner, indicating the ROS scavenging was highly regulated by the interaction of TaVTC2 with NIb. Furthermore, TaVTC2 RNAi plants conferred broad-spectrum disease resistance. Therefore, the data indicate that TaVTC2 recruits WYMV NIb to down-regulate its own enzymatic activity, reducing AsA accumulation to elicit a burst of ROS which confers the resistance to WYMV infection. Thus, a new mechanism of the formation of plant innate immunity was proposed.

[Biomechanical properties of epithelial mesenchymal transition in idiopathic pulmonary fibrosis].

Idiopathic pulmonary fibrosis (IPF) is a progressive scar-forming disease with a high mortality rate that has received widespread attention. Epithelial mesenchymal transition (EMT) is an important part of the pulmonary fibrosis process, and changes in the biomechanical properties of lung tissue have an important impact on it. In this paper, we summarize the changes in the biomechanical microenvironment of lung tissue in IPF-EMT in recent years, and provide a systematic review on the effects of alterations in the mechanical microenvironment in pulmonary fibrosis on the process of EMT, the effects of mechanical factors on the behavior of alveolar epithelial cells in EMT and the biomechanical signaling in EMT, in order to provide new references for the research on the prevention and treatment of IPF.

Promising Approach in the Treatment of Glaucoma Using Nanotechnology and Nanomedicine-Based Systems.

Glaucoma is considered a leading cause of blindness with the human eye being one of the body's most delicate organs. Ocular diseases encompass diverse diseases affecting the anterior and posterior ocular sections, respectively. The human eye's peculiar and exclusive anatomy and physiology continue to pose a significant obstacle to researchers and pharmacologists in the provision of efficient drug delivery. Though several traditional invasive and noninvasive eye therapies exist, including implants, eye drops, and injections, there are still significant complications that arise which may either be their low bioavailability or the grave ocular adverse effects experienced thereafter. On the other hand, new nanoscience technology and nanotechnology serve as a novel approach in ocular disease treatment. In order to interact specifically with ocular tissues and overcome ocular challenges, numerous active molecules have been modified to react with nanocarriers. In the general population of glaucoma patients, disease growth and advancement cannot be contained by decreasing intraocular pressure (IOP), hence a spiking in future research for novel drug delivery systems and target therapeutics. This review focuses on nanotechnology and its therapeutic and diagnostic prospects in ophthalmology, specifically glaucoma. Nanotechnology and nanomedicine history, the human eye anatomy, research frontiers in nanomedicine and nanotechnology, its imaging modal quality, diagnostic and surgical approach, and its possible application in glaucoma will all be further explored below. Particular focus will be on the efficiency and safety of this new therapy and its advances.

Yeast 3',5'-bisphosphate nucleotidase: an affinity tag for protein purification.

Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. Yeast 3',5'-bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3'-phosphoadenosine 5'-phosphate (PAP) with submicromolar Km, which indicated the tight interactions between HAL2 and PAP. In order to explore the feasibility of HAL2 as a protein purification affinity tag, HAL2 was further characterized with PAP as substrate. Results demonstrated that KmPAP and kcatPAP were ∼0.3µM and ∼11s(-)(1), respectively. Kd for PAP was 0.008µM in the presence of Ca(2+). pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd∼8nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca(2+), which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by ethylene glycol tetraacetic acid (EGTA) for elution. A series of expression vectors were further constructed and Escherichia coli adenosine 5'-phosphosulfate kinase (APSK) was prokaryotically expressed, purified and characterized. Ready to use expression vector with eight commonly used restriction enzyme recognition sites in multiple cloning site was subsequently constructed. By comparing with current popular tags, HAL2 was found to be an efficient and economical tag for prokaryotic protein expression and purification.

An Accurate, Rapid and Cost-Effective Method for T-nos Detection Based on CRISPR/Cas12a.

CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase terminator (T-nos) of genetically modified (GM) crops, circumventing the need for expensive instruments and technicians. For enhanced sensitivity and stability of PCR-CRISPR/Cas12a detection, we separately optimised the reaction systems for PCR amplification and CRISPR/Cas12a detection. Eleven samples of soybean samples were assessed to determine the applicability of the PCR-CRISPR/Cas12a method. The method could specifically detect target gene levels as low as 60 copies in the reaction within 50 min. In addition, accurate detection of all 11 samples confirmed the applicability. The method is not limited by large-scale instruments, making it suitable for mass detection of transgenic components in plants in the field. In conclusion, we developed a new, accurate, rapid, and cost-effective method for GM detection.

A Localized CRISPR Assay that Detects Short Nucleic Acid Fragments in Unamplified Genetically Modified Samples.

Detecting short genetically modified (GM) nucleic acid fragments in GM crops and associated products is critically important for the global agriculture industry. Although nucleic acid amplification-based technologies have been widely used for genetically modified organism (GMO) detection, they still struggle to amplify and detect these ultra-short nucleic acid fragments in highly processed products. Here, we used a multiple-CRISPR-derived RNA (crRNA) strategy to detect ultra-short nucleic acid fragments. By combining confinement effects on local concentrations, an amplification-free CRISPR-based short nucleic acid (CRISPRsna) system was established to detect the cauliflower mosaic virus 35S promoter in GM samples. Moreover, we demonstrated assay sensitivity, specificity, and reliability by directly detecting nucleic acid samples from GM crops with a wide genomic range. The CRISPRsna assay avoided possible aerosol contamination from nucleic acid amplification and saved time due to an amplification-free approach. Given that our assay displayed distinct advantages over other technologies in detecting ultra-short nucleic acid fragments, it may have wide applications for detecting GM in highly processed products.

[Fostering the chemistry and biology dual-disciplinary core literacy].

Improving students' comprehensive quality and developing their core literacy are the training objectives of high school subject curriculums, which puts forward new requirements for teachers' professionalism and core literacy. In order to adapt to the high school curriculum reform and the new college entrance examination reform, the Ministry of Education approved the development of high-quality, integrated and master-level high school teachers training pilot program. The aim of this program was to foster a group of dual-disciplinary integrated high school teachers who are suitable for teaching, enjoying teaching and skillful in teaching. How to foster the dual-disciplinary core literacy of normal students through subject curriculum is one of the challenges faced by the pilot program. Following the training objectives of the pilot program "Chemistry and Biological Sciences", we proposed to integrate the dual-disciplinary literacy into four aspects: material concept, scientific thinking, inquiry practice, attitude and responsibility. This was proposed based on analyzing the core literacy of the disciplines of chemistry and biology, and aimed to promote teachers and normal students' understanding and practice of dual-discipline core literacy. With biochemistry course as an example, we further explored and practiced on how to foster the dual-disciplinary core literacy of normal students, aiming to provide reference for the reform of other courses included in the dual-disciplinary integrated programs.

Development and Application of a Visual Duck Meat Detection Strategy for Molecular Diagnosis of Duck-Derived Components.

To make meat adulteration detection systems faster, simpler and more efficient, we established a duck-derived meat rapid detection Recombinase Polymerase Amplification (dRPA) method by using interleukin 2 (IL-2) from nuclear genomic DNA as the target gene to design specific primers. We tested the dRPA detection system by comparing its sensitivity and specificity using real-time fluorescent PCR technology. By adjusting the ratio of reagents, this method shortens the time of DNA extraction and visualizes results in combination with colloidal gold immunoassay strips. Our results demonstrate that this dRPA method could specifically detect duck-derived components with a sensitivity of up to 23 copies/µL and the accuracy of the results is consistent with real-time fluorescent PCR. Additionally, dRPA can detect at least 1% of the duck meat content by mixing beef and mutton with duck meat in different proportions, which was verified by spot-check market samples. These results can be visualized with colloidal gold immunoassay strips with the same accuracy as real-time fluorescent RPA. dRPA can complete detection within 30 min, which shortens existing detection time and quickly visualizes the detection results on-site. This lays the groundwork for future large-scale standardized duck origin detection.

Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

The human estrogen sulfotransferase: a half-site reactive enzyme.

The affinity of 17beta-estradiol (E(2)) for the estrogen receptor is weakened beyond the point of physiological relevance by the transfer of the sulfuryl moiety (-SO(3)) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 3'-hydroxyl of E(2). The mechanism of this transfer reaction, catalyzed by estrogen sulfotransferase (EST), is investigated here in detail. The enzyme (a dimer of identical protomers) presents a clear example of half-sites reactivity--only one of the subunits of the dimer produces product during the catalytic cycle. This is the first example of half-sites reactivity in the sulfotransferase family. A burst of product, with an amplitude that corresponds to one-half of the available active sites, reveals that the mechanism is rate-limited by product release. The equilibrium constant governing interconversion of the substrate (E.PAPS.E(2)) and product (E.PAP.E(2)S) central complexes was determined and is strongly biased toward product (K(eq int) approximately 49). Slow product release allows the interconversion of the central complexes to approach equilibrium, with the result that K(eq int) becomes nearly linearly coupled to K(m) and contributes a factor of approximately 30 to the steady-state affinity of the enzyme for substrate. Typical of its family, estrogen sulfotransferase is partially k(cat)-inhibited by its acceptor substrate, E(2). This inhibition does not influence the burst kinetics and thus occurs after formation of the product central complex, a finding consistent with the slow escape of PAP from the nonreactive E.PAP.E(2) complex.

Mevalonate analogues as substrates of enzymes in the isoprenoid biosynthetic pathway of Streptococcus pneumoniae.

Survival of the human pathogen Streptococcus pneumoniae requires a functional mevalonate pathway, which produces isopentenyl diphosphate, the essential building block of isoprenoids. Flux through this pathway appears to be regulated at the mevalonate kinase (MK) step, which is strongly feedback-inhibited by diphosphomevalonate (DPM), the penultimate compound in the pathway. The human mevalonate pathway is not regulated by DPM, making the bacterial pathway an attractive antibiotic target. Since DPM has poor drug characteristics, being highly charged, we propose to use unphosphorylated, cell-permeable prodrugs based on mevalonate that will be phosphorylated in turn by MK and phosphomevalonate kinase (PMK) to generate the active compound in situ. To test the limits of this approach, we synthesized a series of C(3)-substituted mevalonate analogues to probe the steric and electronic requirements of the MK and PMK active sites. MK and PMK accepted substrates with up to two additional carbons, showing a preference for small substituents. This result establishes the feasibility of using a prodrug strategy for DPM-based antibiotics in S. pneumoniae and identified several analogues to be tested as inhibitors of MK. Among the substrates accepted by both enzymes were cyclopropyl, vinyl, and ethynyl mevalonate analogues that, when diphosphorylated, might be mechanism-based inactivators of the next enzyme in the pathway, diphosphomevalonate decarboxylase.

Diffusional channeling in the sulfate-activating complex: combined continuum modeling and coarse-grained brownian dynamics studies.

Enzymes required for sulfur metabolism have been suggested to gain efficiency by restricted diffusion (i.e., channeling) of an intermediate APS(2-) between active sites. This article describes modeling of the whole channeling process by numerical solution of the Smoluchowski diffusion equation, as well as by coarse-grained Brownian dynamics. The results suggest that electrostatics plays an essential role in the APS(2-) channeling. Furthermore, with coarse-grained Brownian dynamics, the substrate channeling process has been studied with reactions in multiple active sites. Our simulations provide a bridge for numerical modeling with Brownian dynamics to simulate the complicated reaction and diffusion and raise important questions relating to the electrostatically mediated substrate channeling in vitro, in situ, and in vivo.

A Rice CPYC-Type Glutaredoxin OsGRX20 in Protection against Bacterial Blight, Methyl Viologen and Salt Stresses.

Glutaredoxins (GRXs) belong to the antioxidants involved in the cellular stress responses. In spite of the identification 48 GRX genes in rice genomes, the biological functions of most of them remain unknown. Especially, the biological roles of members of GRX family in disease resistance are still lacking. Our proteomic analysis found that OsGRX20 increased by 2.7-fold after infection by bacterial blight. In this study, we isolated and characterized the full-length nucleotide sequences of the rice OsGRX20 gene, which encodes a GRX family protein with CPFC active site of CPYC-type class. OsGRX20 protein was localized in nucleus and cytosol, and its transcripts were expressed predominantly in leaves. Several stress- and hormone-related motifs putatively acting as regulatory elements were found in the OsGRX20 promoter. Real-time quantitative PCR analysis indicated that OsGRX20 was expressed at a significantly higher level in leaves of a resistant or tolerant rice genotype, Yongjing 50A, than in a sensitive genotype, Xiushui 11, exposed to bacterial blight, methyl viologen, heat, and cold. Its expression could be induced by salt, PEG-6000, 2,4-D, salicylic acid, jasmonic acid, and abscisic acid treatments in Yongjing 50A. Overexpression of OsGRX20 in rice Xiushui 11 significantly enhanced its resistance to bacterial blight attack, and tolerance to methyl viologen and salt stresses. In contrast, interference of OsGRX20 in Yongjing 50A led to increased susceptibility to bacterial blight, methyl viologen and salt stresses. OsGRX20 restrained accumulation of superoxide radicals in aerial tissue during methyl viologen treatment. Consistently, alterations in OsGRX20 expression affect the ascorbate/dehydroascorbate ratio and the abundance of transcripts encoding four reactive oxygen species scavenging enzymes after methyl viologen-induced stress. Our results demonstrate that OsGRX20 functioned as a positive regulator in rice tolerance to multiple stresses, which may be of significant use in the genetic improvement of rice resistance.

An affinity-based aqueous two-phase mixed micellar system and its purification of Yeast 3',5'-bisphosphate nucleotidase.

Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system. The addition of HM-EO can result in formation of the micellar network in the micelle-rich phase, making the phase separation easier and stabler. In addition, the extractive performance of ATPMS was enhanced due to the existence of the mixed micelles composed by HM-EO and Cu(II)-chelated TX. It was found in the partitioning experiments that the hexahistidine-tagged Yeast 3',5'-bisphosphate nucleotidase (YND) was selectively extracted into the micelle-rich phase, while the histidine-poor proteins (BSA and lysozyme) remained in the micelle-poor phase. Finally, HM-EO/TX-Cu(II) was used directly to process the fermentation broth. The target protein, YND could be recovered from the cell lysate with a recovery yield of 49.23% and purification factor of 2.63. The results indicated that the new affinity-based HM-EO/TX-Cu(II) system had high partitioning performance which is promising for effectively separation of the histidine-tagged proteins.

A Class II small heat shock protein OsHsp18.0 plays positive roles in both biotic and abiotic defense responses in rice.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating diseases of rice. However, the molecular mechanism underpinning the Xoo resistance of rice is still not fully understood. Here, we report that a class II small heat shock protein gene, OsHsp18.0, whose expression was differentially induced between a resistant and a susceptible variety in response to Xoo infection, plays positive roles in both biotic and abiotic resistance. The molecular chaperone activity of OsHsp18.0 was confirmed by a bacterium-expressed glutathione S-transferase fusion protein. Overexpression of OsHsp18.0 in a susceptible rice variety significantly enhanced its resistance to multiple Xoo strains, whereas silencing of OsHsp18.0 in a resistant variety drastically increased its susceptibility. The enhanced Xoo resistance in OsHsp18.0-overexpressing lines was positively correlated with the sensitized salicylic acid-dependent defense responses. In addition to disease resistance, the OsHsp18.0 overexpressing and silencing lines exhibited enhanced and reduced tolerance, respectively, to heat and salt treatments. The subcellular localization study revealed that the green fluorescent protein-OsHsp18.0 was enriched on the nuclear envelope, suggesting a potential role of OsHsp18.0 in the nucleo-cytoplasmic trafficking. Together, our results reveal that the rice OsHsp18.0 is a positive regulator in both biotic and abiotic defense responses.

Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria.

The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells.

A laboratory exercise for visible gel filtration chromatography using fluorescent proteins.

Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified, and used in a laboratory exercise to intuitively demonstrate GFC. Different bands, corresponding to RFP, RFP-CFP (RC), YFP-RFP-YFP (YRY), and pyruvate kinase II-GFP (PKG) were well separated on a Superdex 200 column from a 0.5-mL sample. Increasing the sample volume and changing the chromatographic resin to Sephadex G-100 resulted in lower resolution separation. Students enjoyed identifying combinations of colored proteins and found this exercise helpful for understanding the factors that affect GFC resolution.

The role of water channel proteins and nitric oxide signaling in rice seed germination.

Previous studies have demonstrated the possible role of several aquaporins in seed germination. But systematic investigation of the role of aquaporin family members in this process is lacking. Here, the developmental regulation of plasma membrane intrinsic protein (PIP) expression throughout germination and post-germination processes in rice embryos was analyzed. The expression patterns of the PIPs suggest these aquaporins play different roles in seed germination and seedling growth. Partial silencing of the water channel genes, OsPIP1;1 and OsPIP1;3, reduced seed germination while over-expression of OsPIP1;3 promoted seed germination under water-stress conditions. Moreover, spatial expression analysis indicates that OsPIP1;3 is expressed predominantly in embryo during seed germination. Our data also revealed that the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), promoted seed germination; furthermore, the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibited germination and reduced the stimulative effects of SNP and GSNO on rice germination. Exogenous NO stimulated the transcription of OsPIP1;1, OsPIP1;2, OsPIP1;3 and OsPIP2;8 in germinating seeds. These results suggest that water channels play an important role in seed germination, acting, at least partly, in response to the NO signaling pathway.

Channeling in sulfate activating complexes.

The synthesis of activated sulfate (adenosine 5'-phosphosulfate, APS) and inorganic pyrophosphate from ATP and SO4 is remarkably unfavorable: K(eq) approximately 10(-8) under presumed, near-physiological conditions. Consequently, ATP sulfurylases, which catalyze APS synthesis, suffer approximately 10(8)-fold losses in catalytic efficiency in the forward (APS-synthesis) versus reverse reaction. Losses of this magnitude place this catalyst at risk of being unable to supply its nutrients to the cell in a timely fashion. ATP sulfurylase domains are often embedded in multifunctional complexes that are capable of also catalyzing the second of two steps in the sulfate activation pathway: the phosphorylation of APS to produce PAPS (3'-phosphoadenosine 5'-phosphosulfate). The colocalization of these activities in a single scaffold suggests that evolution might have worked around the inefficiency problem by fashioning a system capable of transferring APS directly between the active sites of the complex, thereby avoiding the solution-phase energetics. For these reasons, representatives from each of the three types of sulfate activating complex (SAC) [Homo sapiens (type I); Mycobacterium tuberculosis (type II); and Rhodobacter sphaeroides (type III)] were tested for the ability to channel APS. A channeling assay that optically detects solution-phase APS was devised with APS reductase from M. tuberculosis, a previously uncharacterized enzyme. Channeling was not detected in two of the three types of SAC; however, the type III SAC channels with high efficiency. Structural models of type III reveal a 75 A-long channel that interconnects active-site pairs in the complex and that opens and closes in response to occupancy of those sites.