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N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m6A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Intraoperative radiotherapy (IORT) with low-energy X-rays (30-50 KV) is an innovative technique that can be used both for accelerated partial breast irradiation (APBI) and intraoperative boosting in patients affected by breast cancer. Immediately after tumor resection the tumor bed can be treated with low-distance X-rays by a single high dose. Whereas often a geographic miss in covering the boost target occurs with external beam boost radiotherapy (EBRT), the purpose of IORT is to cover the tumor bed safely. This report will focus on the feasibility and technical aspects of the Intrabeam device and will summarize our experience with side effects and local control., Materials and Methods: Between February 2002 and June 2003 57 breast cancer patients, all eligible for breast conserving surgery (BCS), were treated at the Mannheim Medical Center with IORT using the mobile X-ray system Intrabeam. The patient population in this feasibility study was not homogeneous consisting of 49 patients with primary stage I or II breast cancer, seven with local recurrence after previous EBRT and one with a second primary in a previously irradiated breast. The selection criteria for referral for IORT included tumor size, tumor cavity size, margin status and absence of an extensive intraductal component. The previously irradiated patients with local recurrences and 16 others received IORT as single modality. In all other cases IORT was followed by EBRT with a total dose of 46 Gy in 2-Gy fractions. The intraoperatively delivered dose after tumor resection was 20 Gy prescribed to the applicator surface. EBRT was delivered with a standard two-tangential-field technique using linear accelerators with 6- or 18-MV photons. Patients were assessed every three months by their radiation oncologist or surgeon during the first year after treatment and every six months thereafter. Breast ultrasound for follow-up was done every six months and mammographies once yearly. Acute side effects were scored according to the CTC/EORTC score and late side effects according to the Lent-Soma classification., Results: Twenty-four patients received IORT only; eight patients because they had received previous radiotherapy, 16 because of a very favorable risk profile or their own preference. Thirty-three patients with tumor sizes between 1 and 30 mm and no risk factors were treated by IORT as a boost followed by EBRT. The Intrabeam system was used for IORT. The Intrabeam source produces 30-50 KV X-rays and the prescribed dose is delivered in an isotropic dose distribution around spherical applicators. Treatment time ranged between 20 and 48 minutes. No severe acute side effects or complications were observed during the first postoperative days or after 12 months. One local recurrence occurred 10 months after surgery plus IORT followed by EBRT. In two patients distant metastases were diagnosed shortly after BCS., Discussion: IORT with the Intrabeam system is a feasible method to deliver a single high radiation dose to breast cancer patients. As a preliminary boost it has the advantage of reducing the EBRT course by 1.5 weeks, and as APBI it might be a promising tool for patients with a low risk of recurrence. The treatment is well tolerated and does not cause greater damage than the expected late reaction in normal tissue.

The catalytic activities of 4 glycosidases (hyaluronate-4-glycanohydrolase (EC 3.2.1.35), beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30), beta-glucuronidase (EC 3.2.1.31), alpha-L-iduronidase (EC 3.2.1.76)), of the arylsulphatases A and B (EC 3.1.6.1) and of the protease cathepsin D (EC 3.4.23.5) were measured in extracts from hepatocytes and non-parenchymal cells and in serum during the development of thioacetamide-induced rat liver fibrosis (22 weeks). In non-parenchymal liver cells the catalytic activities of beta-N-acetyl-D-glucosaminidase, beta-glucuronidase, alpha-L-iduronidase and cathepsin D were increased significantly during chronic liver damage, but that of hyaluronate-4-glycanohydrolase was reduced by 40 to 65% during the period of application of thioacetamide. The catalytic activities of the arylsulphatases were lowered by 65% compared to control values in the 12th week but with advancing liver damage the catalytic activities returned to nearly normal values. Parenchymal cells of rats, which had been liver-damaged for 6 months, contained strongly elevated activities of beta-glucuronidase, beta-N-acetyl-D-glucosaminidase, arylsulphatases A and B, and cathepsin D but only slightly increased activities of hyaluronate-4-glycanohydrolase and alpha-L-iduronidase, respectively. In the serum of liver-damaged rats the activity of alpha-L-iduronidase was strongly elevated, while that of N-acetyl-beta-D-glucosaminidase was only slightly increased. The activities of beta-glucuronidase and of arylsulphatases A and B were decreased during the whole period of treatment. The catalytic functions of hyaluronate-4-glycanohydrolase and of cathepsin D, respectively, were decreased initially, but both enzyme activities were elevated during the more advanced stages of long term thioacetamide treatment.

Cornea cells were isolated from bovine corneae after collagenase treatment. Subcellular fragments were fractionated by density gradient centrifugation. The density gradient run was monitored by determination of the marker enzyme activities for mitochondria, plasma membranes, lysosomes and endoplasmatic reticulum, of the enzyme activities involved in keratan sulfate synthesis and of the protein content. The fractions were further investigated by electron microscopy. Two membrane fractions with keratan sulfate-synthesizing activity (UDP-N-acetylglucosamine:keratan-N-acetylglucosaminyl-transferase, UDPgalactose:keratan galactosyltransferase and keratan sulfotransferase) were detected: a heavy fraction separated from the other organells investigated and a light fraction exhibiting the same density as plasma membranes. The activities of the three enzymes were found in the same density gradient fractions with a similar distribution pattern between the fractions, which suggests a joint localization of these 3 enzymes at the same intracellular sites.