The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

Author Correction: The Apostasia genome and the evolution of orchids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells.

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

Ligand-Controlled Cobalt-Catalyzed Regio-, Enantio-, and Diastereoselective Oxyheterocyclic Alkene Hydroalkylation.

Metal-hydride-catalyzed alkene hydroalkylation has been developed as an efficient method for C(sp3)-C(sp3) coupling with broad substrate availability and high functional group compatibility. However, auxiliary groups, a conjugated group or a chelation-directing group, are commonly required to attain high regio- and enantioselectivities. Herein, we reported a ligand-controlled cobalt-hydride-catalyzed regio-, enantio-, and diastereoselective oxyheterocyclic alkene hydroalkylation without chelation-directing groups. This reaction enables the hydroalkylation of conjugated and unconjugated oxyheterocyclic alkenes to deliver C2- or C3-alkylated tetrahydrofuran or tetrahydropyran in uniformly good yields and with high regio- and enantioselectivities. In addition, hydroalkylation of C2-substituted 2,5-dihydrofuran resulted in the simultaneous construction of 1,3-distereocenters, providing convenient access to polysubstituted tetrahydrofuran with multiple enantioenriched C(sp3) centers.

Magneto-Structural Correlation of Five-Coordinate Trigonal Bipyramidal High Spin Cobalt(II) Complexes.

Here, we combined magnetometry, multi-frequency electronic paramagnetic resonance, and wave function based ab initio calculations to investigate magnetic properties of two high spin Co(II) complexes Co(BDPRP) (BDPRP=2,6-bis((2-(S)-di(4-R)phenylhydroxylmethyl-1-pyrrolidi-nyl)methyl)pyridine, R=H for 8; R=tBu for 9). Complexes 8 and 9 featuring effective D3h symmetry were found to possess D=24.0 and 32.0 cm-1, respectively, in their S=3/2 ground states of 1 e ' ' d x z / y z 4 1 e ' d x y / x 2 - y 2 2 1 a 1 ' d z 2 1 ${{\left(1{{\rm e}}^{{\rm { {^\prime}}}{\rm { {^\prime}}}}\right({d}_{xz/yz}\left)\right)}^{4}{\left(1{{\rm e}}^{{\rm { {^\prime}}}}\right({d}_{{xy/{x}^{2}-y}^{2}}\left)\right)}^{2}{\left(1{{\rm a}}_{1}^{{\rm { {^\prime}}}}\right({d}_{{z}^{2}}\left)\right)}^{1}}$ . Ligand field analyses revealed that the low-lying d-d excited states make either positive or vanishing contributions to D. Hence, total positive D values were measured for 8 and 9, as well as related D3h high spin Co(II) complexes. In contrast, negative D values are usually observed for C3v congeners. In-depth analyses suggested that lowering symmetry from D3h to C3v induces orbital mixing between 1 e d x z / y z ${1{\rm e}\left({d}_{xz/yz}\right)}$ and 2 e d x y / x 2 - y 2 ${2{\rm e}\left({d}_{{xy/{x}^{2}-y}^{2}}\right)}$ and admixes excited state 4 A 2 1 e → 2 e ${{}^{4}{{\rm A}}_{2}\left(1e\to 2e\right)}$ into the ground state. Both factors turn the total D value progressively negative with the increasing distance (δ) of the Co(II) center out of the equatorial plane. Therefore, δ determines the sign and magnitude of final D values of five-coordinate trigonal bipyramidal S=3/2 Co(II) complexes as measured for a series of such species with varying δ.

A Mechanistic Spectrum of O-H Bond Cleavage Observed for Reactions of Phenols with a Manganese Superoxo Complex.

Reaction of a rare and well-characterized MnIII-superoxo species, Mn(BDPBrP)(O2⋅) (1, H2BDPBrP=2,6-bis((2-(S)-di(4-bromo)phenylhydroxylmethyl-1-pyrrolidinyl)methyl)pyridine), with 4-dimethylaminophenol at -80 °C proceeds via concerted proton electron transfer (CPET) to produce a MnIII-hydroperoxo complex, Mn(BDPBrP)(OOH) (2), alongside 4-dimethylaminophenoxy radical; whereas, upon treatment with 4-nitrophenol, complex 1 undergoes a proton transfer process to afford a MnIV-hydroperoxo complex, [Mn(BDPBrP)(OOH)]+ (3). Intriguingly, the reactions of 1 with 4-chlorophenol and 4-methoxyphenol follow two routes of CPET and sequential proton and electron transfer to furnish complex 2 in the end. UV-vis and EPR spectroscopic studies coupled with DFT calculations provided support for this wide mechanistic spectrum of activating various phenol O-H bonds by a single MnIII-superoxo complex, 1.

Role of n-DAMO in Mitigating Methane Emissions from Intertidal Wetlands Is Regulated by Saltmarsh Vegetations.

Coastal wetlands are hotspots for methane (CH4) production, reducing their potential for global warming mitigation. Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) plays a crucial role in bridging carbon and nitrogen cycles, contributing significantly to CH4 consumption. However, the role of n-DAMO in reducing CH4 emissions in coastal wetlands is poorly understood. Here, the ecological functions of the n-DAMO process in different saltmarsh vegetation habitats as well as bare mudflats were quantified, and the underlying microbial mechanisms were explored. Results showed that n-DAMO rates were significantly higher in vegetated habitats (Scirpus mariqueter and Spartina alterniflora) than those in bare mudflats (P < 0.05), leading to an enhanced contribution to CH4 consumption. Compared with other habitats, the contribution of n-DAMO to the total anaerobic CH4 oxidation was significantly lower in the Phragmites australis wetland (15.0%), where the anaerobic CH4 oxidation was primarily driven by ferric iron (Fe3+). Genetic and statistical analyses suggested that the different roles of n-DAMO in various saltmarsh wetlands may be related to divergent n-DAMO microbial communities as well as environmental parameters such as sediment pH and total organic carbon. This study provides an important scientific basis for a more accurate estimation of the role of coastal wetlands in mitigating climate change.

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans.

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, ß-galactoside-binding proteins, bind to various gram-negative bacterial LPS, which display ß-galactoside-containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1ß production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis-a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Weak gene-gene interaction facilitates the evolution of gene expression plasticity.

BACKGROUND: Individual organisms may exhibit phenotypic plasticity when they acclimate to different conditions. Such plastic responses may facilitate or constrain the adaptation of their descendant populations to new environments, complicating their evolutionary trajectories beyond the genetic blueprint. Intriguingly, phenotypic plasticity itself can evolve in terms of its direction and magnitude during adaptation. However, we know little about what determines the evolution of phenotypic plasticity, including gene expression plasticity. Recent laboratory-based studies suggest dominance of reversing gene expression plasticity-plastic responses that move the levels of gene expression away from the new optima. Nevertheless, evidence from natural populations is still limited. RESULTS: Here, we studied gene expression plasticity and its evolution in the montane and lowland populations of an elevationally widespread songbird-the Rufous-capped Babbler (Cyanoderma ruficeps)-with reciprocal transplant experiments and transcriptomic analyses; we set common gardens at altitudes close to these populations' native ranges. We confirmed the prevalence of reversing plasticity in genes associated with altitudinal adaptation. Interestingly, we found a positive relationship between magnitude and degree of evolution in gene expression plasticity, which was pertinent to not only adaptation-associated genes but also the whole transcriptomes from multiple tissues. Furthermore, we revealed that genes with weaker expressional interactions with other genes tended to exhibit stronger plasticity and higher degree of plasticity evolution, which explains the positive magnitude-evolution relationship. CONCLUSIONS: Our experimental evidence demonstrates that species may initiate their adaptation to new habitats with genes exhibiting strong expression plasticity. We also highlight the role of expression interdependence among genes in regulating the magnitude and evolution of expression plasticity. This study illuminates how the evolution of phenotypic plasticity in gene expression facilitates the adaptation of species to challenging environments in nature.

Free Dermal Fat Grafting: A Novel Technique for the Correction of Nasolabial Folds During Facelift Surgery.

BACKGROUND: Traditional facelift surgery does not behave well in the correction of nasolabial folds, which is a common clinical problem and needed to be improved. OBJECTIVES: To investigate the effect of free dermal fat grafting during facelift surgery for the treatment of nasolabial folds. METHODS: This prospective cohort study involved 80 patients with moderate to severe nasolabial folds and facial skin dermatolysis. Fifty of them underwent facelift surgery combined with free dermal fat grafting, and 30 of them underwent traditional facelift surgery. These patients were followed up 2 months, 6 months, and 1 year after the surgery to evaluate the effect. RESULTS: The difference in Wrinkle Severity Rating Scale (WSRS) scores, assessed at each follow-up, between the patients who underwent and did not undergo free dermal fat grafting during facelift surgery, was statistically significant. For patients who underwent free dermal fat grafting during facelift surgery, the WSRS scores assessed at 2 months, 6 months, and 1 year after the surgery were significantly different from those before the surgery. The analytic results of FACE-Q indicated a high level of overall satisfaction rate. No major complications were recorded. CONCLUSIONS: Free dermal fat as a filler for nasolabial folds can achieve excellent therapeutic effect. The combination of facelift surgery with free dermal fat grafting for the treatment of nasolabial folds can provide very good long-term results and a high patient satisfaction rate for patients with symptoms of facial aging such as facial dermatolysis, obvious wrinkles, and deep nasolabial folds.

An Immunomodulatory Biomimetic Single-Atomic Nanozyme for Biofilm Wound Healing Management.

Nanozyme-driven catalytic antibacterial therapy has become a promising modality for bacterial biofilm infections. However, current catalytic therapy of biofilm wounds is severely limited by insufficient catalytic efficiency, excessive inflammation, and deep tissue infection. Drawing from the homing mechanism of natural macrophages, herein, a hollow mesoporous biomimetic single-atomic nanozyme (SAN) is fabricated to actively target inflamed parts, suppress inflammatory factors, and eliminate deeply organized bacteria for enhance biofilm eradication. In the formulation, this biomimetic nanozyme (Co@SAHSs@IL-4@RCM) consists of IL-4-loaded cobalt SANs-embedded hollow sphere encapsulate by RAW 264.7 cell membrane (RCM). Upon accumulation at the infected sites through the specific receptors of RCM, Co@SAHS catalyze the conversion of hydrogen peroxide into hydroxyl radicals and are further amplify by NIR-II photothermal effect and glutathione depletion to permeate and destroy biofilm structure. This behavior subsequently causes the dissociation of RCM shell and the ensuing release of IL-4 that can reprogram macrophages, enabling suppression of oxidative injury and tissue inflammation. The work paves the way to engineer alternative "all-in-one" SANs with an immunomodulatory ability and offers novel insights into the design of bioinspired materials.

Genomic architecture underlying morphological and physiological adaptation to high elevation in a songbird.

Organisms often acquire physiological and morphological modifications to conquer ecological challenges when colonizing new environments which lead to their adaptive evolution. However, deciphering the genomic mechanism of ecological adaptation is difficult because ecological environments are often too complex for straightforward interpretation. Thus, we examined the adaptation of a widespread songbird-the rufous-capped babbler (Cyanoderma ruficeps)-to a relatively simple system: distinct environments across elevational gradients on the mountainous island of Taiwan. We focused on the genomic sequences of 43 birds from five populations to show that the Taiwan group split from its sister group in mainland China around 1-2 million years ago (Ma) and colonized the montane habitats of Taiwan at least twice around 0.03-0.22 Ma. The montane and lowland Taiwan populations diverged with gene flow between them, suggesting strong selection associated with different elevations. We found that the montane babblers had smaller beaks than the lowland ones, consistent with Allen's rule, and identified candidate genes-COL9A1 and SOX11-underlying the beak size changes. We also found that altitudinally divergent mutations were mostly located in noncoding regions and tended to accumulate in chromosomal inversions and autosomes. The altitudinally divergent mutations might regulate genes related to haematopoietic, metabolic, immune, auditory and vision functions, as well as cerebrum morphology and plumage development. The results reveal the genomic bases of morphological and physiological adaptation in this species to the low temperature, hypoxia and high UV light environment at high elevation. These findings improve our understanding of how ecological adaptation drives population divergence from the perspective of genomic architecture.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Automatic control method for freeform surface shapes.

Designing freeform optics with high degrees of freedom can improve their optical performances; however, there are high requirements for controlling the surface shapes of such optics. Optical designers need to add constraints to the optimization process and make repeated adjustments to ensure the manufacturability of these shapes; this process is cumbersome and relies heavily on the experience of the designer. In this study, an automatic control method for freeform surface shapes is proposed. By adding an outer loop to the optimization process, the principal curvature and sag departure of the sampling points are gradually controlled during the optimization cycle based on the system requirements and surface evaluation results. The method was implemented in CODE V and successfully applied to a design example in freeform prism optics.

Involvement of free-space optics in Raman distributed temperature sensing.

This Letter demonstrates the successful use of free-space optics (FSO) as a transition channel for an air segment in transmitting Raman backscattering signals for distributed temperature sensing (DTS). A barrier-free air segment link shaped by an FSO is part of the Raman-based DTS (RDTS) fiber optic transmission route. For this plan, the FSO enables delivery of the RDTS's pulse with the low-loss transmission over the air segment while also returning to the RDTS the varied Raman backscattered signals from the probing temperature variations for signal interpretation. The difference between various temperatures sensed and the referential air temperature remains nearly the same before and after passing the FSO. The viability of this technology provides a crucial basis for tackling the high expense of installing and repairing DTS cables and the challenges associated with doing so owing to topographical restrictions.

Y3+@CdTe quantum dot nanoprobe as a fluorescence signal enhancement sensing platform for the visualization of norfloxacin.

Quinolone antibiotics (norfloxacin) pose a serious threat to animal and human health due to their misuse and difficulty in being broken down in surface water and food. Rapid and effective detection of norfloxacin (NOR) is essential for environmental testing and ecosystems. In this study, yttrium was coordinated with mercaptopropionic acid (MPA)-modified CdTe quantum dots (QDs) to obtain a novel fluorescence sensor Y3+@CdTe QDs for the sensitive detection of NOR. NOR can bind to Y3+ to form a complex (NOR-Y3+). This complex enhances the luminescence of NOR and blue-shifts to 423 nm. The fluorescence intensity of NOR-Y3+ at 423 nm (I423) gradually increased with increasing NOR concentration; meanwhile, the fluorescence intensity of CdTe QDs at 634 nm (I634) gradually decreased due to aggregation induction. The ratio of I423 to I634 was used for the quantitative determination of NOR. The linear range of the constructed fluorescent probes was from 1.0 to 150.0 µM, with a detection limit of 31.8 nM. CdTe QDs act as a red fluorescent background, and with the addition of NOR, the color of the system transitions from red to purple and finally blue. This method was rapid (immediate) and visual, providing a simple analysis of various actual samples (tap water, lake water, honey, milk and human serum) for NOR.

Fe-N hollow mesoporous carbon spheres with high oxidase-like activity for sensitive detection of alkaline phosphatase.

Due to the vital role of alkaline phosphatase (ALP) in clinical diagnoses and biomedical research, a sensitive and selective detection method for ALP activity is of considerable importance. Herein, a facile and sensitive colorimetric assay for the detection of ALP activity was developed based on Fe-N hollow mesoporous carbon spheres (Fe-N HMCS). Fe-N HMCS were synthesized by a practical one-pot method with aminophenol/formaldehyde (APF) resin as the carbon/nitrogen precursor, silica as the template and iron phthalocyanine (FePC) as the iron source. Thanks to the highly dispersed Fe-N active sites, Fe-N HMCS exhibited exceptional oxidase-like activity. In the presence of dissolved oxygen, Fe-N HMCS were able to effectively convert colorless 3,3',5,5'-tetramethylbenzidine (TMB) into oxidized TMB (oxTMB) with blue color, while the reducing agent of ascorbic acid (AA) inhibited the color reaction. Based on this fact, an indirect and sensitive colorimetric sensing method was developed to detect alkaline phosphatase (ALP) with the assistance of the substrate L-ascorbate 2-phosphate (AAP). This ALP biosensor exhibited a linear range of 1-30 U L-1 and a limit of detection (LOD) of 0.42 U L-1 in standard solutions. In addition, this method was applied to detect ALP activity in human serum with satisfactory results. This work offers a positive reference for the reasonable excavation of transition metal-N carbon compounds in ALP-extended sensing applications.

Dynamics of Benthic Nitrate Reduction Pathways and Associated Microbial Communities Responding to the Development of Seasonal Deoxygenation in a Coastal Mariculture Zone.

Intensive mariculture activities result in eutrophication and enhance coastal deoxygenation. Deoxygenation profoundly influences nitrate reduction processes and further the fate of nitrogen (N) in coastal systems. Herein, 15N isotope labeling, real-time PCR, and high-throughput sequencing techniques were jointly used to investigate the participation and seasonal dynamics of sediment nitrate reduction pathways and the succession of functional microbial communities during the development of seasonal deoxygenation in a coastal aquaculture zone. Denitrification dominated benthic nitrate reduction (46.26-80.91%). Both denitrification and dissimilatory nitrate reduction to ammonium were significantly enhanced by summer deoxygenation (dissolved oxygen levels fell to 2.94 ± 0.28 mg L-1), while anammox remained unchanged. The abundance of the nitrous oxide reductase gene nosZ increased during deoxygenation. The community of the nosZ gene was sensitive to deoxygenation, with Azospirillum and Ruegeria accounting for the majority. Pelobacter was overwhelming in the nrfA gene (encoding dissimilatory nitrite reductase) community, which was less affected by deoxygenation. The variations of benthic nitrate reduction processes were driven by bottom water oxygen combined with temperature, chlorophyll a, and microbial gene abundances and community compositions. Our results implicated that seasonal oxygen-deficient zones could be substantial N sinks of coastal ecosystems and important for N balance. Effective management measures need to be developed to avoid further exacerbation of coastal deoxygenation and maintain the sustainable development of mariculture.

The Apostasia genome and the evolution of orchids.

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

Enhancing the production of a heterologous Trametes laccase (LacA) by replacement of the major cellulase CBH1 in Trichoderma reesei.

The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi. ONE-SENTENCE SUMMARY: The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.