Anion-Vacancy Modified WSSe Nanosheets on 3D Cross-Networked Porous Carbon Skeleton for Non-Aqueous Sodium-Based Dual-Ion Storage.

Sodium-based dual-ion batteries (SDIBs) have become a new type of energy storage device with great application value because of their high operating voltage, high energy density, and low cost. However, transition-metal dichalcogenide (TMD) anodes show unsatisfactory Na+ electrochemical performance owing to the low intrinsic conductivity and inferior ion transport kinetics. Here, an elaborate design is developed to prepare a composite of WSSe nanosheets supported on a 3D cross-networked porous carbon skeleton (WSSe@CPCS), which possesses en-rich anion vacancies and WSSe with expanded inter-layer spacing, as well as an interconnected porous structure. As a result, the WSSe@CPCS anode for sodium-ion batteries (SIBs) exhibits preeminent reversible capacities, excellent cycle stability, and superior rate capability. The systematic electrochemical kinetic analysis and density functional theory results further show that the effect of anion vacancies and CPCS synergistically enhances the conductivity and reduces charge transfer resistance, thus making a great contribution to fast reaction kinetics. Finally, the implementations of the WSSe@CPCS anode in progressive SIB and DIB full-cell configurations exhibit satisfactory performance, which reveals their widely practical application. This research will provide an exciting approach to designing advanced defect-structured tungsten-based TMD materials for SIBs, DIBs, and even a broad range of energy storage.

Preparation of Chitosan/ß-Cyclodextrin Composite Membrane and Its Adsorption Mechanism for Proteins.

A significant portion of the protein in food waste will contaminate the water. The chitosan/modified ß-cyclodextrin (CS/ß-CDP) composite membranes were prepared for the adsorption of bovine serum albumin (BSA) in this work to solve the problem of poor adsorption protein performance and easy disintegration by a pure chitosan membrane. A thorough investigation was conducted into the effects of the preparation conditions (the mass ratio of CS and ß-CDP, preparation temperature, and glutaraldehyde addition) and adsorption conditions (temperature and pH) on the created CS/ß-CDP composite membrane. The physical and chemical properties of pure CS membrane and CS/ß-CDP composite membrane were investigated. The results showed that CS/ß-CDP composite membrane has better tensile strength, elongation at break, Young's modulus, contact angle properties, and lower swelling degree. The physicochemical and morphological attributes of composite membranes before and after the adsorption of BSA were characterized by SEM, FT-IR, and XRD. The results showed that the CS/ß-CDP composite membrane adsorbed BSA by both physical and chemical mechanisms, and the adsorption isotherm, kinetics, and thermodynamic experiments further confirmed its adsorption mechanism. As a result, the CS/ß-CDP composite membrane of absorbing BSA was successfully fabricated, demonstrating the potential application prospect in environmental protection.

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation.

The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitroIMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

Effect of fumonisin B1 on oxidative stress and gene expression alteration of nutrient transporters in porcine intestinal cells.

Fumonisin B1 (FB1 ) is a common environmental mycotoxin produced by molds such as Fusarium verticillioides. The toxin poses health risks to domestic animals, including pigs, through FB1 -contaminanted feed. However, the cytotoxicity of FB1 to porcine intestines has not been fully analyzed. In the present study, the effects of FB1 on oxidative stress and nutrient transporter-associated genes of the porcine intestinal IPEC-J2 cells were explored. FB1 decreased IPEC-J2 proliferation but did not trigger reactive oxygen species (ROS) overproduction. Meanwhile, FB1 reduced the expression levels of the transporters l-type amino acid transporter-1 (y+ LAT1), solute carrier family 7 member 1 (SLC7A1), solute carrier family 1 member 5 (ASCT2), and excitatory amino acid carrier 1 (EAAC1); in addition, FB1 reduced the levels of the fatty acid transporters long-chain fatty acid transport protein 1 (FATP1) and long-chain fatty acid transport protein 4 (FATP4) as well as glucose transporters Na+ /glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2). FB1 stimulation increased the expression levels of peptide transporter peptide transporter 1 (PepT1) and metal ion transport-related gene zinc transporter 1 (ZNT1). Moreover, metal ion transporter divalent metal transporter 1 (DMT1) expression was depressed by a higher dosage of FB1 . The data indicate that FB1 results in aberrant expression of nutrient transporters in IPEC-J2 cells, thereby exerting its toxicity even though it fails to exert ROS-dependent oxidative stress.

Sirtuin 3 improves fatty acid metabolism in response to high nonesterified fatty acids in calf hepatocytes by modulating gene expression.

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and ß-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.

Fumonisin B1 damages the barrier functions of porcine intestinal epithelial cells in vitro.

Fumonisins (Fums) are mycotoxins widely distributed in crops and feed, and ingestion of Fums-contaminated crops is harmful to animal health. The purpose of this study is to explore the effect of Fum B1 (FB1 ) on barrier functions of porcine intestinal epithelial cells, IPEC-J2, to clarify the intestinal toxicity of Fums in pigs. The results showed that the persistent treatment of FB1 significantly decreased the viability of IPEC-J2. Moreover, the expressions of Claudin 1, Occludin, Zonula Occluden-1 (ZO-1) on the messenger RNA (mRNA), and protein levels and MUC1 on the mRNA level were significantly inhibited after FB1 treatment, while the mRNA relative expression level of MUC2 was clearly increased. FB1 also enhanced the monolayer cell permeability of IPEC-J2. Importantly, FB1 promoted the expression of phosphorylated extracellular regulated protein kinase (p-ERK1/2 ). These data suggest that long-term treatment of FB1 can suppress IPEC-J2 proliferation, damage tight junctions of IPEC-J2, and regulate expression of mucins to induce the damage of barrier functions of porcine intestinal epithelial cells, which may be associated with the ERK1/2 phosphorylation pathway.

Effects of Quercetin on Proliferation and H2O2-Induced Apoptosis of Intestinal Porcine Enterocyte Cells.

Weanling stress and toxicosis, which are harmful to the health of pigs' intestines, are associated with oxidative stress. Quercetin (Que) is a polyphenolic compound that shows good anti-cancer, anti-inflammation and anti-oxidation effects. This study aimed to elaborate whether or not Que promotes IPEC-J2 (intestinal porcine enterocyte cells) proliferation and protects IPEC-J2 from oxidative damage. Thus, we examined the effects of Que on proliferation and H2O2-induced apoptosis in IPEC-J2. The results showed that Que increased IPEC-J2 viabililty, propelled cells from G1 phase into S phase and down-regulated gene levels of P27 and P21, respectively. Besides, H2O2-induced cell damage was alleviated by Que after different exposure times, and Que depressed apoptosis rate, reactive oxygen species (ROS) level and percentage of G1 phase cells and elevated the percentage of cells in G2 phase and S phase and mitochondrial membrane potential (Δψm) after IPEC-J2 exposure to H2O2. Meanwhile, Que reduced the value of Bax/Bcl-2 in H2O2 exposed cells. In low-degree oxidative damage cells, lipid peroxidation product malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were increased. In turn, Que could reverse the change of MDA content and SOD activity in low-degree damage cells. Nevertheless, catalase (CAT) activity was not changed in IPEC-J2 incubated with Que under low-degree damage conditions. Interestingly, relative expressive levels of the proteins claudin-1 and occludin were not altered under low-degree damage conditions, but Que could improve claudin-1 and occludin levels, slightly. This research indicates that Que can be greatly beneficial for intestinal porcine enterocyte cell proliferation and it protects intestinal porcine enterocyte cells from oxidation-induced apoptosis, and could be used as a potential feed additive for porcine intestinal health against pathogenic factor-induced oxidative damages and apoptosis.

Evidence of natural co-infection with PCV2b subtypes in vivo.

Porcine circovirus type 2 (PCV2) is the causative pathogen of porcine circovirus-associated diseases (PCVAD). This virus evolves mostly through point mutations and genome recombination between different PCV2 genotypes (e.g. PCV2a and PCV2b), as has been confirmed in swine herds. In the current work, the complete PCV2 genome sequences of 69 clones derived from various tissues (lymph node, spleen and lung,) of an infected individual, were subjected to phylogenetic and alignment analyses. The results not only demonstrate co-infection with distinct PCV2b subtypes (e.g. 1B and 1C) in the same animal, but also highlight another mechanism of evolution - diverse point mutations acquired during immune evasion by this virus.

Combined index finger pollicization with an anterolateral thigh flap for thumb reconstruction.

A severe crush injury to the hand is devastating to patients. Under conditions in which the crush force is too great, the digits are not viable candidates for replantation. We present two cases in which the patients suffered from loss of the thumb ray at the first carpometacarpal joint and skin defect at the radial side. The tendons, radial nerve and metacarpal bone of the index finger were injured, and the second metacarpal head was retained. There was a comminuted fracture of the trapezium in both patients. The treatment protocols consisted of the index finger pollicization and the free anterolateral thigh flap transfer. The procedure was performed in a single operation. The new thumb is able to provide a stable post for pinch and grip after six months follow-up. Both patients were satisfied with the function and appearance of the reconstructed thumb.

Preparation, physicochemical and biological evaluation of chitosan Pleurotus ostreatus polysaccharides active films for food packaging.

The aim of this study was to create CS-POP composite films by blending Pleurotus ostreatus stalk polysaccharides (POP) and chitosan (CS). The effects of adding different concentrations (0 %, 0.25 %, 0.5 %, 0.75 %, and 1 %) of POP on the mechanical, barrier, and optical properties of the CS films were investigated. When the POP content is at 0.5 %, the tensile strength of the composite film reaches its maximum value at 13.691 MPa, showing a significant improvement compared to the tensile strength of the pure CS film. The structure of the CS and CS-POP composite films was characterized by FT-IR spectroscopy, XRD, TGA and SEM. The results indicate that due to the interaction between the two types of CS and POP, the formation of Schiff base, and the intermolecular hydrogen bonds between CS and POP, the addition of POP to CS films can result in a smoother and more stable crystalline structure in the composite film. The CS-POP composite films exhibited enhanced antioxidant and antibacterial activity compared to the CS films alone, with the highest DPPH scavenging activity of 72.43 %. The composite films also showed significant inhibitory effects on the growth of E. coli.

Investigating the physicochemical characteristics and importance of insoluble dietary fiber extracted from legumes: An in-depth study on its biological functions.

Legumes are widely appreciated for their abundant reserves of insoluble dietary fiber, which are characterized by their high fiber content and diverse bioactive compounds. Insoluble dietary fiber in leguminous crops is primarily localized in the structural cell walls and outer integument and exhibits strong hydrophilic properties that enable water absorption and volumetric expansion, resulting in increased food bulk and viscosity. This contributes to enhanced satiety and accelerated gastrointestinal transit. The benefits of legume insoluble dietary fiber extend to its notable antioxidant, anti-inflammatory, and anti-cancer properties, as well as its ability to modulate the composition of the intestinal microbiota, promoting the growth of beneficial bacteria while suppressing the proliferation of harmful pathogens, thereby promoting optimal intestinal health. It is highly valued as a valuable thickening agent, stabilizer, and emulsifier, contributing to the texture and stability of a wide range of food products.

Advancements in modifying insoluble dietary fiber: Exploring the microstructure, physicochemical properties, biological activity, and applications in food industry-A review.

Recent research has primarily focused on strategies for modifying insoluble dietary fiber (IDF) to enhance its performance and functionality. IDF is obtained from various inexpensive sources and can be manipulated to alter its biological effects, making it possible to revolutionize food processing and nutrition. In this review, multiple IDF modification techniques are thoroughly examined and discussed, with particular emphasis on the resulting changes in the physicochemical properties, biological activities, and microstructure of the fiber. An extensive overview of the practical applications of modified IDF in food processing is provided. Our study aims to raise awareness about the vast possibilities presented by modified IDF and encourage further exploration and utilization of this field in the realm of food production.

Recent advancements in chitosan-based intelligent food freshness indicators: Categorization, advantages, and applications.

With an increasing emphasis on food safety and public health, there is an ongoing effort to develop reliable, non-invasive methods to assess the freshness of diverse food products. Chitosan-based food freshness indicators, leveraging properties such as biocompatibility, biodegradability, non-toxicity, and high stability, offer an innovative approach for real-time monitoring of food quality during storage and transportation. This review introduces intelligent food freshness indicators, specifically those utilizing pH-sensitive dyes like anthocyanins, curcumin, alizarin, shikonin, and betacyanin. It highlights the benefits of chitosan-based intelligent food freshness indicators, emphasizing improvements in barrier and mechanical properties, antibacterial activity, and composite film solubility. The application of these indicators in the food industry is then explored, alongside a concise overview of chitosan's limitations. The paper concludes by discussing the challenges and potential areas for future research in the development of intelligent food freshness indicators using chitosan. Thus, chitosan-based smart food preservation indicators represent an innovative approach to providing real-time data for monitoring food quality, offering valuable insights to both customers and retailers, and playing a pivotal role in advancing the food industry.

Preparation of chitosan/Enoki mushroom foot polysaccharide composite cling film and its application in blueberry preservation.

In this study, the composite cling film was prepared by solution casting method using chitosan and golden mushroom foot polysaccharide as substrates, and the structure and physicochemical indexes of the composite cling film were characterized by Fourier infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. The results showed that compared with single chitosan film, the composite cling film has better mechanical properties and antioxidant properties, and the barrier of UV light and water vapor is also stronger. Due to its high nutritional value, blueberry has a short shelf life due to its thin skin and poor storage resistance. Therefore, in this study, blueberry was used as the object of freshness preservation, and the single chitosan film group and the uncovered group were used as controls, and the weight loss, total bacterial colony, decay rate, respiration intensity, malondialdehyde content, hardness, soluble solids, titratable acid, anthocyanin content, and VC content of blueberry were used as freshness preservation indexes for experiments. The comprehensive results showed that the freshness preservation effect of the composite film group was significantly higher than that of the control group, with better antibacterial properties, antioxidant properties, etc., which could effectively delay fruit decay and deterioration, thus prolonging the shelf life, and thus the chitosan/Enoki mushroom foot polysaccharide composite preservation film has a high potential as a new freshness preservation material for blueberry.

Nickel sulfide/nickel phosphide heterostructures anchored on porous carbon nanosheets with rapid electron/ion transport dynamics for sodium-ion half/full batteries.

Nickel-based materials have been extensively deemed as promising anodes for sodium-ion batteries (SIBs) owing to their superior capacity. Unfortunately, the rational design of electrodes as well as long-term cycling performance remains a thorny challenge due to the huge irreversible volume change during the charge/discharge process. Herein, the heterostructured ultrafine nickel sulfide/nickel phosphide (NiS/Ni2P) nanoparticles closely attached to the interconnected porous carbon sheets (NiS/Ni2P@C) are designed by facile hydrothermal and annealing methods. The NiS/Ni2P heterostructure promotes ion/electron transport, thus accelerating the electrochemical reaction kinetics benefited from the built-in electric field effect. Moreover, the interconnected porous carbon sheets offer rapid electron migration and excellent electronic conductivity, while releasing the volume variance during Na+ intercalation and deintercalation, guaranteeing superior structural stability. As expected, the NiS/Ni2P@C electrode exhibits a high reversible specific capacity of 344 mAh g-1 at 0.1 A g-1 and great rate stability. Significantly, the implementation of NiS/Ni2P@C//Na3(VPO4)2F3 SIB full cell configuration exhibits relatively satisfactory cycle performance, which suggests its widely practical application. This research will develop an effective method for constructing heterostructured hybrids for electrochemical energy storage.

Dorsalis pedis arterialized venous flap for hand and foot reconstruction.

OBJECTIVE: To report the results of repair of skin defects in the extremities with arterialized venous flap harvested from the lateral aspect of the dorsum of the foot. METHODS: Six cases of skin and soft tissue defects over the foot and hands were resurfaced by free arterialized venous flaps, including five patients with skin defects of the hands, and one with defects at the dorsum of the foot. The flaps were harvested from the lateral aspect of the dorsum of the foot with the sizes ranging from 2 cm multiply 5.5 cm to 6 cm multiply 11 cm. Two veins at the proximal margin of the flap were retained, one of which was anastomosed to a recipient bed artery to provide arterial inflow and the other was anastomosed to a recipient bed vein for venous outflow. RESULTS: All flaps demonstrated mild edema and survived completely. Blisters appeared on four flaps. Using this technique, we achieved good functional and cosmetic results in this series. CONCLUSIONS: Dorsalis pedis arterialized venous flap with rich vascular communications could enhance peripheral perfusion and decrease congestion of venous flaps, thereby improves reliability and utility for extremity reconstruction.

Effect of Fumonisin B1 on Proliferation and Apoptosis of Intestinal Porcine Epithelial Cells.

Fumonisin B1 (FB1), which is a mycotoxin produced by Fusarium moniliforme and Fusarium rotarum, has a number of toxic effects in animals. Moldy feed containing FB1 can damage the intestine. In this study, we used intestinal porcine epithelial cells (IPEC-J2) as an in vitro model to explore the effects of FB1 on cell cycle and apoptosis. The results showed that IPEC-J2 cells treated with 10, 20, and 40 µg/mL FB1 for 48 h experienced different degrees of damage manifested as decreases in cell number and viability, as well as cell shrinkage and floating. In addition, FB1 reduced cell proliferation and the mRNA and protein expression of proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 2 (CDK2), CDK4, cyclinD1, and cyclinE1. FB1 blocked the cell cycle in the G1 phase. FB1 also induced mitochondrial pathway apoptosis, reduced mitochondrial membrane potential, and promoted mRNA and protein expression of Caspase3, Caspase9, and Bax. The findings suggest that FB1 can induce IPEC-J2 cell damage, block the cell cycle, and promote cell apoptosis.

Pseudorabies in pig industry of China: Epidemiology in pigs and practitioner awareness.

Pseudorabies virus (PRV) is widely prevalent in China, which can transmit from pigs to other mammals. Moreover, a PRV variant isolated from an acute human encephalitis case was documented recently. It is imperative to investigate PRV epidemiology in pigs, the knowledge regarding pseudorabies (PR) and self-protection behaviors upon working among relevant practitioners including pig farmers, pig cutters, and pork salesman. In the present study, 18,812 pig serum samples and 1,634 tissue samples were collected from Hunan Province during the period of 2020 to 2021 for detecting the presence of PRV gE-special antibody and nucleic acids, respectively. Meanwhile, we conducted a questionnaire survey about PR among these practitioners in China. The results showed that nearly 9% (1,840/20,192) pigs from 161 collected sites (20.17%, 161/797) were seropositive for PRV-gE antibody. Though only 2.33% tissue samples were positive for PRV nucleic acids, all the representative PRV strains were variant. It was learned that most practitioners were frequently injured when working, the injured sites mainly included hand and foot. Among the three transmission routes of PRV, the aerosol transmission route was often overlooked. Moreover, the workers lacked self-protection awareness and were poor conscious about PRV and its potential threat to humans. All the results demonstrate that PRV remains widely spread in pig populations, while the potential threats of PRV in pig industry receive less attention, suggesting that targeted educational programs to these people should be performed.

Treatment of degloving injury of three fingers with an anterolateral thigh flap.

The degloving injuries of the digits and palm remain a persistent challenge. We used an antero- lateral thigh flap to treat an 18-year-old, right-handed male worker with degloving injuries of the index, middle and ring fingers. The flap was designated to wrap the entire circumference of three fingers sustaining degloving injury and to form mitten-hand. The total lengths of the distal phalanxes of three fingers were retained almost complete. The donor defect was covered with split-thickness skin graft. Three months after the first operation, roentgenograms revealed terminal phalanxex resorption in three injured fingers, and the surgical syndactyly between the middle and ring finger was separated at the same time. One month later, the syndactyly between the index and middle fingers was also separated. Good coverage of the soft tissue defects with good function and appearance was achieved. Therefore, we considered that the length of the degloved finger could be preserved using free flap.