Genome-wide single nucleotide polymorphism array and whole-genome sequencing reveal the inbreeding progression of Banna minipig inbred line.

Inbred pigs are promising animal models for biomedical research and xenotransplantation. Established in 1980, the Banna minipig inbred (BMI) line originated from a sow and its own male offspring. It was selected from a small backcountry minority Lahu village, where records show that no other pig breed has ever been introduced. During the inbreeding process, we perfomed extreme inbreeding over 23 consecutive generations using full-sibling or parent-offspring mating. In order to investigate the inbreeding effects in BMI pigs across generations over the past 40 years, in this study we conducted a genome-wide SNP genotyping of the last 10 generations, representing generations 14-23. In total, we genotyped 57,746 SNPs, corresponding to an average decrease in heterozygosity rate of 0.0078 per generation. Furthermore, we were only able to identify 18,216 polymorphic loci with a MAF larger than 0.05, which is substantially lower than the values in previous reports on other pig breeds. In addition, we sequenced the genome of the first pig in the twenty-third generation (inbreeding coefficient 99.28%) to an average coverage of 12.4× to evaluate at the genome level the impact of advanced inbreeding. ROH analysis indicates that BMI pigs have longer ROHs than Wuzhishan and Duroc pigs. Those long ROH regions in BMI pigs are enriched for distinct functions compared with the highly polymorphic regions. Our study reveals a genome-wide allele diversity loss during the progress of inbreeding in BMI pigs and characterizes ROH and polymorphic regions as a result of inbreeding. Overall, our results indicate the successful establishment of the BMI line, which paves the way for further in-depth studies.

Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 µM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer.

A novel tool for intralymphatic injection: the modified glass hypodermic needle.

The use of contrast lymphangiography is a relatively new technique in lymphatic anatomical research, employed as a tool for evaluating lymphatic anatomy in fresh cadaveric specimens. With the use of microsurgical techniques to cannulate lymphatics, contrast media can enable the use of lymphangiography for evaluating lymphatic anatomy. However, the ability to cannulate lymphatics with diameters that are less than the smallest commercially available needles has been a significant limitation, and indeed a challenge. The smallest commercially available hypodermic needles have been 30-gauge needles, with 0.3 mm outer diameters. The lymphatics for cannulation in our studies are of the order of 0.1 mm, and other options have been required. We describe a novel technique for cannulating lymphatic vessels, creating a modified glass hypodermic needle. We have shown that these glass needles can be made with accuracy to diameters as low as 0.01 mm. Although 0.1 mm glass needles are the more commonly utilized in most dissections, we can now accurately create these glass needles to any caliber between 0.01 mm and 0.1 mm, based on the predicted lymphatic anatomy.

Advanced imaging technologies for mapping cadaveric lymphatic anatomy: magnetic resonance and computed tomography lymphangiography.

Lymphatic anatomy has become increasingly clinically important as surgical techniques evolve for investigating and treating cancer metastases. However, due to limited anatomical techniques available, research in this field has been insufficient. The techniques of computed tomography (CT) and magnetic resonance (MR) lymphangiography have not been described previously in the imaging of cadaveric lymphatic anatomy. This preliminary work describes the feasibility of these advanced imaging technologies for imaging lymphatic anatomy. A single, fresh cadaveric lower limb underwent lymphatic dissection and cannulation utilizing microsurgical techniques. Contrast materials for both CT and MR studies were chosen based on their suitability for subsequent clinical use, and imaging was undertaken with a view to mapping lymphatic anatomy. Microdissection studies were compared with imaging findings in each case. Both MR-based and CT-based contrast media in current clinical use were found to be suitable for demonstrating cadaveric lymphatic anatomy upon direct intralymphatic injection. MR lymphangiography and CT lymphangiography are feasible modalities for cadaveric anatomical research for lymphatic anatomy. Future studies including refinements in scanning techniques may offer these technologies to the clinical setting.

The lymphatics of the skin filled by a dermal backflow: an observation in a scarred cadaver leg.

There have been few photographic studies done on lymphatics of human skin with previous images obtained by indirect dye injection into the dermis. We have developed a direct injection technique for investigating the lymphatic system in human adult cadavers and used this to investigate superficial lymphatics of the legs in a fresh human cadaver. We found an unusual observation in a skin graft scarred leg when the radio-opaque mixture injected into a lymph collecting vessel in the dorsal foot filled the skin lymphatics in the mid-lateral pretibial region. Further radiological investigation revealed that the dermal back flow was associated with a blockage of the lymph collecting vessel within the skin graft scar. We also found tracer transport through a circuitous pathway from the blocked collecting vessel to an adjacent intact collecting vessel. The transilluminated image of the skin demonstrated a three dimensional polygon of lymph capillaries and precollecting lymphatics in the dermis.

The angiosomes of the head and neck: anatomic study and clinical applications.

The angiosome concept was introduced over a decade ago by Taylor and Palmer, whereby the body was considered to be composed anatomically of multiple three-dimensional composite blocks of tissue supplied by particular source arteries. Since then, detailed studies of the forearm and leg have been examined by Taylor and his coworkers. This study focuses on another region--the head and neck. Six fresh head and neck cadaver specimens were examined after infusion with a radio-opaque lead oxide mixture and correlated with over 24 previous body studies. The vascular anatomy of the skin, superficial musculoaponeurotic system (SMAS), muscles, brain, dura, and bone was examined. Each layer was painstakingly removed, photographed, labeled, and mapped to the respective arteries and veins. A radiologic subtraction technique was used to allow successive layers to be compared. This information was then scanned into a computer, analyzed, color coded, and labeled, thereby producing a three-dimensional study of the head and neck region to identify the respective angiosomes. As in previous detailed examinations of the leg and forearm, the angiosomes were found to be connected usually within tissues, such as muscle, skin, specialized organs or glands, rather than between the tissues. The muscles usually had vessels of two or more angiosomes supplying them and fell into three major groups based on the similarity of their arterial supply. In some areas, the midline anastomoses were rich, especially in the integument of the scalp, forehead, and lips. In other regions, the midline vascular connections were poor, especially in the tongue and palate. No fewer than 13 angiosomes of the head and neck, supplied by the branches of the external carotid, internal carotid, and subclavian arteries, have been defined, mapping their three-dimensional territories in the skin, the deep soft tissues, and the bones. Although most angiosomes spanned between skin and bone, three territories, those of the vertebral, lingual, and ascending pharyngeal vessels, were confined to the deep tissues without cutaneous representation. Finally, this study provides additional data for the surgeon to help plan safer incisions and better reconstructive flap procedures. It also gives information that may help explain the etiology and treatment of head and neck arteriovenous vascular malformations.

Angiosomes of the leg: anatomic study and clinical implications.

In 1987, Taylor and Palmer introduced the angiosome concept. This anatomical study defined the three-dimensional vascular territories supplied by source arteries and veins to each tissue layer between the skin and the bone. This report, however, was an overview investigation and did not study each region of the body in fine detail. In 1996, Inoue and Taylor studied the angiosomes of the forearm in much greater detail. They showed, among other findings, that the zone between the angiosomes, formed by reduced caliber (choke) vessels or similar caliber (true) anastomotic arteries, occurred usually within tissues, especially the muscles, not between them. This study focuses on the same region in the lower limb to draw a comparison and to fill certain voids in our knowledge--the leg. Twelve lower limbs from fresh cadavers were investigated over a 2-year period after perfusing each with a mixture containing radio-opaque lead oxide. The anatomy of the arterial supply to the skin, the muscles, and the periosteum of the bones of the leg was examined. The contribution to each tissue was defined by dissection, by metal clip tagging of vessels, by radiography, and by mapping the branches with colored pins, coded to match the respective source arteries. A subtraction technique was used to study the muscles whereby the bones of the limb were replaced with radiolucent balloons to obtain an unobscured picture of the vasculature of the leg. The muscles were then segregated one by one from the muscle mass and x-rayed again. Next, cross-section studies were made in two legs to complete the three-dimensional picture, tracing the branches from the source arteries to each layer. Finally, the contribution to each tissue from the popliteal, sural, anterior tibial, posterior tibial, and peroneal vessels were color coded to match these source arteries, thus defining the angiosomes of the leg. Results, as in the forearm, showed that in most cases the connections between adjacent angiosomes occurred within tissues, not between them. The skin, the bones, and most muscles received branches from two or more angiosomes, thus revealing one of the important anastomotic pathways through which the circulation is reconstituted when a source artery is interrupted by disease or trauma. Notably, however, the muscles of the anterior compartment of the leg were supplied from one angiosome. This finding, coupled with the anatomy of the rigid fascial compartments of the leg, helps explain the variable clinical pictures and syndromes seen in cases in which the circulation is compromised or interrupted. Finally, this anatomical study adds further information to help design or redesign flaps in the leg for local or free transfer. Similarly, the information reveals the pathways through which the supply to the remaining tissues is reconstituted when one of the source arteries is harvested with a free flap, especially when multiple tissues are included in the transplant.