Mid-trimester residual cervical length and the risk of preterm birth in pregnancies after abdominal radical trachelectomy: a retrospective analysis.

OBJECTIVE: To investigate the association between mid-trimester residual cervical length (CL) and the risk of preterm birth in pregnancies after abdominal radical trachelectomy (RT). DESIGN: Retrospective cohort study. SETTING: University hospital. POPULATION: A total of 33 deliveries after 22 weeks' gestation in 30 women who underwent abdominal RT including prophylactic cervical cerclage and perinatal care between January 2002 and May 2016. METHODS: The association between mid-trimester residual CL (the distance between the cerclage and the external cervical os) and gestational age at delivery was investigated. Receiver-operating characteristics (ROC) curve analysis was performed to estimate the optimal cut-off values of the mid-trimester residual CL for the prediction of preterm birth. MAIN OUTCOME MEASURES: Preterm birth before 34 weeks' gestation. RESULTS: Mid-trimester residual CL showed a significant correlation with gestational age at delivery (r = 0.36, P < 0.05). There was a significant difference in residual CL between women who did and those who did not give birth before 34 weeks (P < 0.05). Mid-trimester residual CL < 13 mm was a good predictor of birth before 34 weeks, with a sensitivity of 67%, specificity of 75%, positive predictive value of 55% and negative predictive value of 86% (area under ROC curve, 0.75). CONCLUSIONS: Mid-trimester residual CL is significantly correlated with gestational age at delivery. Residual CL assessment could be used to reassure physicians and women that there is only a small chance of preterm birth in pregnancies after abdominal RT. TWEETABLE ABSTRACT: Mid-trimester residual cervical length is a good predictor of preterm birth after radical trachelectomy.

Finances and returns for robotic dairies.

Automatic milking systems (AMS) became commercially available in the early 1990s. These systems provide flexibility and improve the lifestyle of farmers installing them. Because of the larger capital cost per kilogram of milk produced, observational studies in Europe and simulation studies have shown AMS to be less profitable than milking parlor systems, although previous findings are somewhat mixed. Improved performance of newer generations of AMS, better facility design to accommodate cow behavior, and better management of these facilities have the potential to make AMS more profitable. Wage rates are also increasing and sourcing high-quality milking labor is challenging. We developed partial budget simulations to model profitability of AMS compared with parlor systems for 120-, 240-, and 1,500-cow farms. Both the 120-cow and 240-cow AMS were more profitable than the parlor systems. However, the 1,500-cow parlor system was more profitable than the AMS. Breakeven labor analysis of the 1,500-cow system showed that at a wage inflation rate of 1% and a 0.91 kg/d lower milk production with the AMS system, the breakeven labor rate was $27.02/h. If the farm is able to achieve similar milk production between the 2 systems and wage inflation averages 3% over the 30-yr time horizon, the breakeven wage rate drops to $17.11/h. The major management factors that influenced the net annual impact were changes in milking labor cost and milk production. Another significant factor affecting net annual impact was the economic life of the AMS. An economic life of 13 yr or longer was required for an AMS to have a consistently positive net annual impact (depending on milk production per cow and labor cost). For every 227-kg increase in daily milk production per AMS, net annual income increased approximately $4,100. Cost-effective ways to optimize milk per AMS are to minimize attaching and milking times and to optimize milking settings.

Competitiveness of management-intensive grazing dairies in the mid-Atlantic region from 1995 to 2009.

This paper used farm income tax returns (Schedule F) data from 62 dairy farmers who milked 200 cows or fewer in western and central Maryland and southwestern Pennsylvania (hereafter, the mid-Atlantic region) to assess the relative financial performance of management-intensive grazing (MIG) and confinement dairy operations over the 15-yr period from 1995 through 2009. Data were not available from all farmers in all years; on average, the sample analyzed contained 11 MIG farms and 26 confinement farms. Management-intensive grazing operators were more profitable on a per hundredweight, per cow, and per acre basis, and no less profitable on a whole-farm basis. Even though the confinement operators had higher gross income than MIG operators, their expenses exceeded those of MIG operators. Profits of MIG operations were less variable as well, so that MIG operators faced less income risk. Increased reliance on grazing has other benefits as well. Grazing seems to be a much healthier practice for dairy cows. Veterinary, breeding, and medicine costs per cow are much less for cows that are pastured than those raised in confinement systems. Because they are healthier, cows that are grazed can be milked longer (or culled less frequently). As a result, MIG operators have a larger number of higher quality animals for sale (e.g., bred heifers). Management-intensive operations are also less labor intensive. Reductions in crop production and in the time cows spend in the barn led to significant reductions in field work and cleaning operations in the barn. Costs of hired labor were thus substantially lower in MIG operations than in confinement operations. Land requirements likely impose the principal limitation on the size of intensive grazing operations. In the mid-Atlantic, for instance, grazing operations need 1.5 to 2.0 acres of pasture for every dairy cow/calf equivalent to provide sufficient grass to support a dairy operation. Pasture land for MIG operators must be contiguous to the milking parlor and located no farther than a cow can walk to and from twice a day. That requirement likely limits the maximum size of an intensive grazing operation, especially in areas where land prices and rents are high, as they are in much of the mid-Atlantic.

Effect of selective vaccination on a decrease in the rate of hepatitis B virus-positive Japanese first-time blood donors.

The government of Japan started a selective vaccination programme to prevent mother-to-infant infection by hepatitis B virus (HBV) since January 1986. The effect of the programme on first-time blood donors has not been examined in detail. Data of first-time blood donors aged 16-25 years from 1996 to 2007 were extracted from the Japanese Red Cross (JRC) donors' database. Principal component analysis (PCA) was used to visualize the birth-year-dependent group of rate of HBV-positive donors. According to the birth of year, donors were divided into four groups by PCA. After the start of the programme, donors born in 1986-1989 comprised a single group. Before the start of the programme, three groups (1980, 1981-1984 and 1985) were identified. Although a significant time-dependent decrease in the rate of HBV-positive donors was observed before the start of the programme, a significant difference in the rate of HBV-positive donors was observed around the start of the programme by regression analysis for 16-19-year-old first-time blood donors. The selective vaccination programme has been effective to prevent the vertical transmission of HBV from the analysis of first-time blood donors. On the other hand, vaccination of blood donors should be considered to reduce the risk of post-transfusion HBV infection, because the horizontal transmission increases in HBV-positive blood donors.

Superiority of minipool nucleic acid amplification technology for hepatitis B virus over chemiluminescence immunoassay for hepatitis B surface antigen screening.

BACKGROUND AND OBJECTIVES: The Japanese Red Cross (JRC) have developed a fully automated multiplex (MPX) nucleic acid amplification technology (NAT) system for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1). This is used to test serologically negative blood units from volunteer, non-remunerated donors. The system utilizes a 50-sample pool for NAT screening with an input volume of each pool. This results in a significantly higher sensitivity for hepatitis B than that seen with highly sensitive hepatitis B surface antigen (HBsAg) testing. MATERIALS AND METHODS: From 1 February 2000 to 15 October 2001, over 11 million donations, which were serologically negative, were tested using the MPX NAT system. Donations found to be HBV DNA positive were further tested by using the chemiluminescence immunoassay (CLIA). RESULTS: Out of 181 HBV DNA-positive donations, 96 (53%) and 76 (42%) were negative by individual enzyme immunoassay (EIA) and CLIA testing, respectively. CONCLUSIONS: The sensitivity of the 50-sample pool MPX NAT system was higher than that of individual HBsAg screening by CLIA. By adopting this NAT-screening system, the JRC has improved the safety of the blood supply and maintained supply across Japan.

Status of NAT screening for HCV, HIV and HBV: experience in Japan.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. The NAT screening assay using multiplex reagent is time-saving, cost effective, and labour-saving procedure for all blood and blood products including short-shelf life platelets. During the 50-mini-pool NAT screening of serologically negative donations (February 1, 2001-April 30, 2001), we were able to screen out 112 HBV-positive, 25 HCV-positive, and 4 HIV-1 positive units from blood and blood components.

Prenatal diagnosis of midgut volvulus by sonography and magnetic resonance imaging.

We present a case of congenital midgut volvulus detected by prenatal sonography and ultrafast magnetic resonance (MR) imaging. At 34 weeks of gestation, enlarged hyperechogenic loops without peristalsis was identified by sonographic examination. On ultrafast T2-weighted single-shot fast-spin echo MR imaging, enlarged loops exhibited a lower signal intensity than the surrounding bowel loops, suggesting intraluminal hemorrhage. At explorative laparotomy following delivery, midgut volvulus causing hemorrhagic necrosis was found. Combined use of sonography and ultrafast MR imaging is useful to identify fetal midgut volvulus with hemorrhagic change.

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.

Participation of reactive oxygen species in PGF2alpha-induced apoptosis in rat luteal cells.

Prostaglandin F(2alpha) (PGF(2alpha)) is implicated in the process of luteal regression in many species. Treatment of rat luteal tissue with PGF(2alpha) increases the generation of reactive oxygen species. Since reactive oxygen species have been implicated in apoptosis, the present study was undertaken to determine whether reactive oxygen species play a role in the PGF(2alpha)-induced apoptosis of rat luteal cells. Rat luteal cells were loaded with 6-carboxy-2, 7'-dichlorodihydro-fluorescein (CDCFH) diacetate, di (acetomethyl ester), which can be oxidized by reactive oxygen species to yield CDCF, a fluorescent molecule, and the cells were treated with different doses of PGF(2alpha). Incubation with 100 micromol PGF(2alpha) l(-1) induced an increase in CDCF fluorescence (P < 0. 05). Treatment of cells with PGF(2alpha) for 48 h in serum-free medium induced a dose-dependent increase in cell death, and these cells exhibited the morphological characteristics typical of apoptosis, including condensed or fragmented nuclei and fragmentation of internucleosomal DNA. Pretreatment of these cells with ascorbic acid, N,N'-dimethylthiourea, or superoxide dismutase, which acts as an antioxidant or a radical scavenger, prevented the PGF(2alpha)-induced apoptosis. These results demonstrate that PGF(2alpha) produces reactive oxygen species and induces apoptosis in rat luteal cells, indicating that the reactive oxygen species may induce apoptotic cell death during luteolysis.

Power and colour Doppler ultrasonography for the evaluation of the vasculature of the human corpus luteum.

We used power Doppler imaging to examine neovascularization in the corpus luteum (CL) in 12 healthy volunteers. We also investigated whether CL blood flow reflected luteal function. The ratio of the area of vessels in the CL to the area of a sectional plane at the maximum diameter of the CL observed by power Doppler (FA ratio) was used as a quantitative index of the vascularity of the CL. The pulsatility index (PI) was significantly lower in ovarian arteries with CL than without CL (P < 0.05). Changes in ovarian arterial and intra-luteal PI appeared to reflect physiological changes in the vasculature of the CL. There was no correlation between the volume of the CL or the FA ratio and the concentration of progesterone. The pattern of changes in the product of the FA ratio and the CL volume and in the progesterone concentration was similar. The progesterone concentration was positively correlated with this product (r = 0.74, P < 0.01). The product of the FA ratio and the CL volume plateaued during the mid- to late luteal phase, suggesting the presence of functional and structural luteolysis. These findings suggest that colour Doppler ultrasonography, including power Doppler imaging, can detect physiological changes in the blood flow of the ovary in the luteal phase, and may be a useful noninvasive tool for evaluating CL function.

Induction of cytochrome P450 1A2 by musk analogues and other inducing agents in rat liver.

We characterized the inducing effects of two musk analogues, musk xylene and musk ambrette, on phase I and phase II drug-metabolizing enzymes in rat liver and compared their effects with 3-methylcholanthrene, isosafrole and 2(3)-tertbutylhydroxyanisole (BHA) at 0.1 mmol/kg dose level. Musk xylene and isosafrole increased more efficiently the metabolic activation of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to mutagen than that of benzo(a)pyrene. Musk ambrette increased both the activation of Glu-P-1 and benzo(a)pyrene to the same extent. Western blot analyses revealed that musk xylene, musk ambrette, isosafrole and BHA induced more strongly cytochrome P450 1A2 (CYP1A2) in microsomes than CYP1A1. 3-Methylcholanthrene induced CYP1A1 in preference to CYP1A2. On the other hand, all drugs except for 3-methylcholanthrene did not show remarkable increases in phase II enzyme activities, such as DT-diaphorase, glutathione S-transferase and UDP-glucuronyltransferase, at 0.1 mmol/kg dose level. These results show that musk xylene, musk ambrette, isosafrole and BHA at the dose level used in this study possess the potency to induce CYP1A2 without remarkable induction of CYP1A1 and phase II enzyme activities as observed for 3-methylcholanthrene, although they have been considered to induce both phase I and phase II drug-metabolizing enzymes at higher doses.

An unusual profile of musk xylene-induced drug-metabolizing enzymes in rat liver.

We have demonstrated previously that musk xylene, a non-mutagenic carcinogen, is a novel and specific inducer of CYP1A2 in rats (Iwata et al., Biochem Biophys Res Commun 184: 149-153, 1992). In the present study, the effects of musk xylene (50, 100 or 200 mg/kg body weight, i.p., for 5 consecutive days) on both Phase I and Phase II metabolizing enzymes in rat liver were investigated further and more completely. Among the mixed-function oxidases monitored, 7-ethoxycoumarin deethylase and 7-pentoxyresorufin depentylase activities were increased at all dose levels from 1.6- to 1.7-fold and 2.6- to 3.1-fold, respectively. Benzo[a]pyrene hydroxylase activity was increased significantly at only the 200 mg/kg dose level of musk xylene (1.5-fold). Regarding Phase II enzymes, activities of both cytosolic DT-diaphorase and glutathione S-transferase (GST) were increased up to 2.0- to 2.4-fold by musk xylene in a dose-dependent manner. Western blot analysis revealed that the changes in these activities were caused by increases in the amounts of DT-diaphorase and GST Ya subunit. Microsomal UDP-glucoronyltransferase (UDPGT) activity assayed with p-nitrophenol as substrate was increased 1.6- to 2.0-fold. These results show that musk xylene induces both Phase I cytochrome P450 mixed-function oxidase (CYP1A2 specific) and Phase II metabolizing enzyme systems (DT-diaphorase, GST Ya subunit and UDPGT) in rat liver.

Musk xylene is a novel specific inducer of cytochrome P-450IA2.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.

Effect of sorbic acid feeding on peroxisomes and sorboyl-CoA metabolizing enzymes in mouse liver. Selective induction of 2,4-dienoyl-CoA hydratase.

On the basis of the finding that sorbic acid (SA)-induced hepatoma was correlated with the depletion of reduced glutathione (GSH) in mouse liver (Tsuchiya et al., Mutation Res 130: 267-262, 1984), the possible conversion of SA to a metabolite which is reactive with SH-compounds was studied. Sorboyl-CoA was hydrated and then reduced to 3-keto-4-hexenoyl-CoA by the combined actions of mitochondrial hydratase (crotonase) and L-3-hydroxyacyl-CoA dehydrogenase. Upon the addition of GSH or coenzyme A, 3-keto-4-hexenoyl-CoA was nonenzymatically converted to another 3-ketoacyl-CoA derivative, possibly a Michael type adduct, in a time- and concentration-dependent manner. Alternatively, sorboyl-CoA can be reduced by 2,4-dienoyl-CoA reductase and completely beta-oxidized without the generation of 3-keto-4-hexenoyl-CoA. Two-week feeding of mice of 15% SA caused a 2.0-fold induction of peroxisome beta-oxidation in the liver. SA caused a marked induction (3.6-fold) of hydratase toward sorboyl-CoA but a less pronounced induction (1.3-fold) of 2,4-dienoyl-CoA reductase, leading to about a 3-fold elevation in the hydratase: reductase ratio. The elevated ratio was sustained throughout the period of SA feeding up to 12 weeks. Thus, a large amount of SA could be converted to 3-keto-4-hexenoyl-CoA during this period. Oxidative stress caused by a depleted cellular SH-pool together with the induction of peroxisome proliferation by SA-feeding may implicate the mechanism by which non-mutagenic SA caused hepatoma.

Distribution, metabolism, and excretion of musk xylene in rats.

Distribution, metabolism and excretion of musk xylene (MX) were investigated in male Wistar rats. Urinary and fecal excretion accounted for 10 and 75% of the dose (70 mg/kg), respectively, on day 7 after orally administration of 3H-MX to rats. Total residue of radioactivity in tissues on day 7 was less than 2.0% of the administered dose. The highest concentration was found in adipose tissue and the second was in liver. Some metabolites of MX were identified using GC-MS and NMR after purification by column or thin layer chromatography of feces, bile and urine extracts. MX, 2-NH2-MX, 2-Ac-MX, 2-NH2-3-CH2OH-MX, and 2-NH2-5-tert-BuOH-MX were found in feces, bile and urine. 4-NH2-MX and metabolite X were found in feces and urine. 4-NH2-3-CH2OH-MX was found in urine. HO-MX was found in bile. The major route of excretion for MX was the feces via bile. The reduction of the 2-nitro group of MX to the amino group was a key step in metabolism. Further metabolism of 2-NH2-MX may proceed by decreased steric hindrance of functional group.

Prevalence of anti-HCV antibody in blood donors in the Tokyo area.

Prospective studies of posttransfusion hepatitis carried out in the past decade showed that 18.1% of the blood transfusions resulted in non-A non-B hepatitis in Japan. As an approach to the prevention of posttransfusion non-A non-B hepatitis (PTNANB), anti-hepatitis C virus (HCV) positivity was measured in 2,970 blood donations in the Tokyo area, and in 200 children aged between 6 and 15 years. Thirty-four cases were anti-HCV-positive, showing an overall positivity of 1.14%. None of the 200 children younger than 15 years old were positive. Correlation of anti-HCV positivity with the serum ALT levels was observed, but by reducing the accepted ALT levels from 35 Karmen Units (KU) down to 25 KU, it is estimated that 62.5% of the observed PTNANB would still have occurred, and 5.1% of the donated blood could not be used for transfusion. On the other hand, it is estimated that the majority of PTNANB could be prevented, with the loss of 1.14% of donated blood units, using the anti-HCV screening test.

Toluene transposons Tn4651 and Tn4653 are class II transposons.

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.

Characterization of skin-surface lipids from the monkey (Macaca fascicularis).

Skin-surface lipids from the monkey Macaca fascicularis are composed of sterol esters (38%), cholesterol (4%) and two types of wax diesters, identified as Type II (IIa and IIb, 17% and 40%, respectively). Type IIa contained diesters of 1,2-alkanediols esterified with two molecules of long-chain (C14-C34) fatty acids having straight and branched chains. In the diesters IIa, fatty acids shorter than C19 predominated in position 1, and fatty acids longer than C20 predominated in position 2. Type IIb contained diesters of 1,2-alkanediols esterified with C4 and C5 branched-chain fatty acids (predominantly isovaleric acid) at position 1 and long-chain (C14-C27) acids, having straight and branched chains, at position 2. The short-chain acids were converted to 2-nitrophenylhydrazides and analyzed by high-performance liquid chromatography (HPLC). Ammonia chemical ionization (CI)-gas chromatography (GC)-mass spectrometry (MS) resolved the intact diesters IIb into 12 peaks corresponding to molecular weights ranging from 597 to 748, and showed that the molecular species, such as C21-C16-C5 (diol, fatty acid in position 2, fatty acid in position 1), C22-C16-C5 and C23-C16-C5, were prevalent. The fatty acids from both diesters were mostly (greater than 98%) saturated. The 1,2-alkane-diols from both diesters consisted of C16-C26 saturated straight- and branched-chain components. The acyl groups of sterol esters contained 86% C14-C34 branched-chain acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of rose bengal on serum levels of thyroid hormones and thyroid peroxidase activity in male mice.

The thyrotoxic effect of Rose bengal (RB) (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein disodium salt; Food Red No. 105) was examined in male (C57BL/6N X C3H/N) F1 mice. They were given drinking-water containing RB at levels of 0 (control), 0.125 and 0.250% for 2 weeks. The effect resulted in decreases in serum levels of 3,5,3'-triiodothyronine (T3) and thyroxine (T4), and slight increases in serum 3,3',5'-triiodothyronine (rT3) levels and thyroid weight, but no difference in the values for the body-weight gain, serum thyroid stimulating hormone (TSH) levels and thyroid peroxidase (TPO) activities. However, the in vitro inhibitory effect of RB on TPO activity was observed by addition of RB to the TPO-catalyzed guaiacol oxidation. These results suggest that RB might have weak goitrogenic properties, inhibiting the peripheral conversion of T4 to T3 and/or inhibiting TPO to lead a decrease of T4 and T3 formation.