In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E. coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production. We named these inclusion body-associated proteins IbpA and IbpB. The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E. coli gene bank. The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome. Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp. Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes. A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB. A temperature upshift experiment in which we used a wild-type E. coli strain and an isogenic rpoH mutant strain indicated that a 32-containing RNA polymerase is involved in the regulation of expression of these genes. There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level. A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.One of the most widely studied and utilized hosts for heterologous gene expression is the gram-negative bacterium Escherichia coli. Numerous genes encoding prokaryotic, viral, or eukaryotic proteins have been cloned and expressed in E. coli. High-level accumulation of some proteins in E. coli results in the formation of intracellular electron-dense protein aggregates that are referred to as inclusion bodies (17,23,43). In some instances, these aggregates appear to be refractile when they are examined by phase-contrast microscopy (similar to gram-positive endospores); such aggregates have been termed refractile bodies (17). Some examples of cloned proteins which, when they are overexpressed, result in the formation of refractile bodies include E. coli proteins, such as aspartase (28), the sigma subunit of RNA polymerase (11), and penicillin acylase (37), and eukaryotic proteins, such as human immunodeficiency virus reverse transcriptase (21), poliovirus 3C protease (16) human insulin (8), human renin (15, 38), and bovine growth hormone (36).In this paper we describe two 16-kDa proteins that were produced during high-level expression in E. coli of some heterologous proteins, such as human renin, prorenin, and bovine insulin-like growth factor 2 (bIGF-2). The 16-kDa proteins were tightly associated with inclusion bodies which were formed in cells that overproduced these heterologous proteins. The genes encoding the 16-kDa proteins were cloned from the Kohara E. coli gene bank (18) by using degenerate oligonucleotide probes generated from the amino acid sequences of th...

Hepatocellular carcinoma (HCC) or liver cancer is one of the fastest growing cancers in the United States. Current liver ablation methods are thermal-based and share limitations due to the heat sink effect from the blood flow through the highly vascular liver. In this study, we demonstrate the feasibility of using histotripsy for non-invasive liver ablation in the treatment of liver cancer. Histotripsy is a non-thermal ablation method that fractionates soft tissue through the control of acoustic cavitation. Twelve histotripsy lesions ~1cm3 were created in the livers of six pigs through an intact abdomen and chest in vivo. Histotripsy pulses of 10 cycles, 500 Hz pulse repetition frequency (PRF), and 14-17 MPa estimated in situ peak negative pressure were applied to the liver using a 1 MHz therapy transducer. Treatments were performed through 4-6 cm of overlying tissue with 30-50% of the ultrasound pathway covered by the ribcage. Complete fractionation of liver parenchyma was observed with sharp boundaries after 16.7 minute treatments. In addition, two larger volumes of 18 cm3 and 60 cm3 were generated within 60 minutes in two additional pigs. As major vessels and gallbladder have higher mechanical strength and are more resistant to histotripsy, the major hepatic vessels and gallbladder remained intact while the liver surrounding these structures was completely fractionated. This work demonstrates that histotripsy is capable of non-invasively fractionating liver tissue while preserving critical anatomical structures within the liver. Results suggest histotripsy has potential for the non-invasive ablation of liver tumors.

Skeletal elements have a diverse range of shapes and sizes specialized to their various roles including protecting internal organs, locomotion, feeding, hearing, and vocalization. The precise positioning, size, and shape of skeletal elements is therefore critical for their function. During embryonic development, bone forms by endochondral or intramembranous ossification and can arise from the paraxial and lateral plate mesoderm or neural crest. This review describes inductive mechanisms to position and pattern bones within the developing embryo, compares and contrasts the intrinsic vs extrinsic mechanisms of endochondral and intramembranous skeletal development, and details known cellular processes that precisely determine skeletal shape and size. Key cellular mechanisms are employed at distinct stages of ossification, many of which occur in response to mechanical cues (eg, joint formation) or preempting future load‐bearing requirements. Rapid shape changes occur during cellular condensation and template establishment. Specialized cellular behaviors, such as chondrocyte hypertrophy in endochondral bone and secondary cartilage on intramembranous bones, also dramatically change template shape. Once ossification is complete, bone shape undergoes functional adaptation through (re)modeling. We also highlight how alterations in these cellular processes contribute to evolutionary change and how differences in the embryonic origin of bones can influence postnatal bone repair.

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The changes identified appear similar, albeit milder to the changes reported in horses with THO, suggesting that degenerative, rather than infectious causes may underlie the aetiology of THO. Future work should be directed at examining the histopathology of clinical THO cases.