Effect of Sustained Joint Loading on TMJ Disc Nutrient Environment.

The temporomandibular joint (TMJ) disc nutrient environment profoundly affects cell energy metabolism, proliferation, and biosynthesis. Due to technical challenges of in vivo measurements, the human TMJ disc extracellular nutrient environment under load, which depends on metabolic rates, solute diffusion, and disc morphometry, remains unknown. Therefore, the study objective was to predict the TMJ disc nutrient environment under loading conditions using combined experimental and computational modeling approaches. Specifically, glucose consumption and lactate production rates of porcine TMJ discs were measured under varying tissue culture conditions (n = 40 discs), and mechanical strain-dependent glucose and lactate diffusivities were measured using a custom diffusion chamber (n = 6 discs). TMJ anatomy and loading area were obtained from magnetic resonance imaging of healthy human volunteers (n = 11, male, 30 ± 9 y). Using experimentally determined nutrient metabolic rates, solute diffusivities, TMJ anatomy, and loading areas, subject-specific finite element (FE) models were developed to predict the 3-dimensional nutrient profiles in unloaded and loaded TMJ discs (unloaded, 0% strain, 20% strain). From the FE models, glucose, lactate, and oxygen concentration ranges for unloaded healthy human TMJ discs were 0.6 to 4.0 mM, 0.9 to 5.0 mM, and 0% to 6%, respectively, with steep gradients in the anterior and posterior bands. Sustained mechanical loading significantly reduced nutrient levels (P < 0.001), with a critical zone in which cells may die representing approximately 13.5% of the total disc volume. In conclusion, this study experimentally determined TMJ disc metabolic rates, solute diffusivities, and disc morphometry, and through subject-specific FE modeling, revealed critical interactions between mechanical loading and nutrient supply and metabolism for the in vivo human TMJ disc. The results suggest that TMJ disc homeostasis may be vulnerable to pathological loading (e.g., clenching, bruxism), which impedes nutrient supply. Given difficulties associated with direct in vivo measurements, this study provides a new approach to systematically investigate homeostatic and degenerative mechanisms associated with the TMJ disc.

Breast size and breast cancer: a systematic review.

BACKGROUND: There are many known breast cancer risk factors, but traditionally the list has not included breast size. The aim of this study was to synthesize the literature on breast size as a risk factor for breast carcinoma by examining studies addressing this question both directly and indirectly. METHODS: A systematic review was performed searching MEDLINE from 1950 to November 2010, and updated again in February 2014. Literature was sought to assess the relationship between the following variables and breast cancer: 1) breast size; 2) breast reduction; 3) breast augmentation; and 4) prophylactic subcutaneous mastectomy. Findings were summarized and the levels of evidence were assessed. RESULTS: 50 papers were included in the systematic review. Increasing breast size appears to be a risk factor for breast cancer, but studies are limited by their retrospective nature, imperfect size measurement techniques and confounding variables. The evidence is stronger for risk reduction with breast reduction, including prophylactic subcutaneous mastectomy at the extreme. Generally the breast augmentation population has a lower risk of breast cancer than the general population, but it is unclear whether or not this is related to the bias of small breasts in this patient population and the presence of other confounders. CONCLUSIONS: There is direct and indirect evidence that breast size is an important factor in the risk of developing breast cancer. Plastic surgeons are in a unique position to observe this effect. Well-designed prospective studies are required to further assess this risk factor.

Low-level resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein 1 in vitro associated with qacA gene carriage is independent of multidrug efflux pump activity.

Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.

Revising the troublesome stoma: combined abdominal wall recontouring and revision of stomas.

PURPOSE: Despite preoperative siting and maturation of stomas, some patients may have poor stoma function because of redundant pannus, scars, creases, and parastomal or incisional hernias. In these patients, a combined abdominoplasty and stoma revision may be helpful. The object of this manuscript is to report our preliminary results. METHODS: Eight patients (mean age, 48 years; female/male ratio, 7/1) undergoing this procedure are reported. Five patients had inflammatory bowel disease, two had malignancies, and one had multiple sclerosis with bladder and bowel involvement. Patients were contacted at two months to two years follow-up to assess functional outcome and satisfaction with the procedure. RESULTS: Indications for surgery were difficulty maintaining an appliance (n = 7), frequent stool leakage (n = 6), and skin irritation (n = 4). Multiple surgeries (n = 4), skin creases (n = 3), scarring (n = 2), large weight loss (n = 4), and hernias (n = 5) were contributing factors. At surgery five patients had hernias repaired (3 parastomal, 2 incisional), three had stomas resited, and three underwent resections for Crohn's disease. One patient developed a seroma postoperatively. At follow-up six patients were able to maintain an appliance for at least four days, whereas one changed the appliance every three days because of personal preference. None experience stool leakage. All reported a dramatic improvement in body image. CONCLUSION: Combined stoma revision and abdominoplasty can be performed safely and leads to improved functional results and outcome.

An investigation into the changed physiological state of Vibrio bacteria as a survival mechanism in response to cold temperatures and studies on their sensitivity to heating and freezing.

AIMS: To induce pathogenic Vibrio bacteria into a changed physiological state, in response to cold temperatures in sea water, and assess their sensitivity to heating and freezing, as compared with normal cells. METHODS AND RESULTS: Cells of exponential phase Vibrio vulnificus, V. cholerae and V. parahaemolyticus were washed and inoculated into flasks of sea water, which were stored at 20 and 4 degrees C. Cells stored at 20 degrees C could be recovered after 60 d on non-selective agar (heart infusion agar; HIA) and on the selective agar (thiosulphate citrate bile salts agar) which is used in most Vibrio detection methodology. At 4 degrees C cells became non-culturable on both agars over time. The non-culturable cells appeared to be metabolically active and maintained their membrane integrity, whilst undergoing a change in morphology from rod-shaped to coccoid cells. Resuscitation was possible, in some cases, by an upshift in temperature before plating and the addition of catalase to HIA plates was found to increase recovery. Studies were carried out to assess the sensitivity of the non-culturable cells to heating and freezing compared with the normal cells. Vibrio organisms, whether culturable or in the non-culturable form, were not inactivated by freezing to -20 degrees C. Heating studies showed that V. parahaemolyticus was very heat resistant at low temperatures. However, a pasteurization regime of 2 min at 70 degrees C was found to be effective against all three strains. Experiments showed that the non-culturable cells of all three strains were similar in their heat resistance or, in some cases, were more heat sensitive than cells in the normal form. CONCLUSIONS: Cells in the changed physiological form would not be detected in fish or seafood products by the current Vibrio detection methods. Freezing had no effect in reducing cell numbers. Vibrio parahaemolyticus was very heat resistant in the low temperature pasteurization studies. The higher pasteurization regime of 70 degrees C for 2 min was effective against all three pathogens. Non-culturable cells had similar heat sensitivity or were more heat sensitive than cells in the normal state. SIGNIFICANCE OF IMPACT OF THE STUDY: The study has highlighted a need for the development of better Vibrio detection methods. The low temperature pasteurization of oysters, which has been recommended in the USA, would not be adequate against the strain of V. parahaemolyticus used in this study. Heating regimes which were found to control cells in the normal form will also be effective for the control of the cells with changed physiology.

Structural mechanisms of QacR induction and multidrug recognition.

The Staphylococcus aureus multidrug binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by structurally diverse cationic lipophilic drugs. Here, we report the crystal structures of six QacR-drug complexes. Compared to the DNA bound structure, drug binding elicits a coil-to-helix transition that causes induction and creates an expansive multidrug-binding pocket, containing four glutamates and multiple aromatic and polar residues. These structures indicate the presence of separate but linked drug-binding sites within a single protein. This multisite drug-binding mechanism is consonant with studies on multidrug resistance transporters.

The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers.

Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.

A comparison of two methods of infiltration in breast reduction surgery.

The superwet technique has been shown in previous studies to dramatically reduce blood loss in breast reduction surgery, compared with standard infiltration. A retrospective chart review of 303 consecutive patients undergoing bilateral breast reduction surgery was undertaken to demonstrate additional differences in complication rate, operative time, or sponge use in the operating room. In this series, 132 consecutive patients received standard infiltration along incision lines (25 cc per breast of 1:100,000 epinephrine), and 171 patients received superwet infiltration with 240 cc per breast of 1:1,000,000 epinephrine. The average operative time was significantly reduced in the superwet group, from 78.5 minutes to 70.7 minutes (p < 0.01 level). The average number of sponges used intraoperatively was also decreased significantly (p < 0.01), from 26 to 20 sponges. Complication rates were equally low in both groups, demonstrating the safety of the superwet technique. In addition to limiting blood loss, the superwet infiltration effectively reduces operative time and sponge use without increasing complications in breast reduction surgery.

Transcriptional regulation of multidrug efflux pumps in bacteria.

As integral membrane proteins demonstrating an extraordinarily wide substrate range, some degree of regulatory control over the expression of bacterial multidrug-resistance (MDR) transporters is to be expected. Excessive expression could be deleterious, due to direct, physical disruption of membrane integrity, or the unwanted export of essential metabolites, a potential side-effect of their broad substrate specificity. There are limited clues as to the physiological functions of most MDR transporters, but their expression is likely to be up-regulated in response to the presence of natural substrates of these pumps. Thus, it is no surprise that MDR genes are subject to regulation at the local level, consisting of examples of both transcriptional repression and activation by proteins encoded adjacent to that for the transporter. Furthermore, an increasing number of MDR genes have also been found to be controlled by global transcriptional activator proteins.

Staphylococcal multidrug efflux protein QacA.

The QacA multidrug exporter from Staphylococcus aureus mediates resistance to a wide array of monovalent or divalent cationic, lipophilic, antimicrobial compounds. QacA provides resistance to these various compounds via a proton motive force-dependent antiport mechanism that conforms to classical Michaelis-Menten kinetics. Fluorescent transport analyses have demonstrated that this QacA:substrate interaction occurs with high affinity and competition studies have shown that QacA-mediated ethidium export is competitively inhibited by other monovalent cations, and non-competitively inhibited by divalent cations, suggesting that monovalent and divalent cations bind at distinct sites on the QacA protein. The closely related export protein QacB, mediates lower levels of resistance to divalent cations, and lacks a high affinity-binding site for divalent cations. The cell membrane has been identified as the origin of QacA-mediated efflux; substrates are bound and expelled from within this hydrophobic environment. Regulation of qacA expression is achieved via the transacting repressor protein, QacR. QacR belongs to the TetR family of transcriptional repressor proteins, which all possess a helix-turn-helix DNA-binding domain at their N-terminal ends, and have highly divergent C-termini postulated to be involved in the binding of inducing compounds. QacR specifically binds to an inverted repeat, IR1, which has been identified as the qacA operator region, and overlaps the identified promoter sequence for qacA. QacR, like the multidrug export protein whose expression it regulates, has been shown to interact directly with a number of structurally-dissimilar compounds.

The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans.

OX2 (CD200) is a type-1 membrane glycoprotein that contains two immunoglobulin superfamily domains and which is expressed on a variety of lymphoid and non-lymphoid cells in the rat. The recent characterization of a receptor for OX2 (OX2R) on myeloid cells, and the phenotype of an OX2-deficient mouse, suggests that OX2 may regulate myeloid cell activity in anatomically diverse locations. Here we report the tissue distribution of the human homologue of the rat OX2 glycoprotein using a new monoclonal antibody (mAb), OX104, raised against recombinant human OX2. Human OX2 was expressed at the cell surface of thymocytes, B cells, T cells, neurons, kidney glomeruli, tonsil follicles, the syncytiotrophoblast and endothelial cells. This broad, but not ubiquitous, distribution pattern is very similar to that observed in rats, suggesting that OX2 may regulate myeloid cell activity in a variety of tissues in humans.

Down-regulation of the macrophage lineage through interaction with OX2 (CD200).

OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.

Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.

The OX2 membrane glycoprotein (CD200) is expressed on a broad range of tissues including lymphoid cells, neurons, and endothelium. We report the characterization of an OX2 receptor (OX2R) that is a novel protein restricted to cells of the myeloid lineage. OX2 and its receptor are both cell surface glycoproteins containing two immunoglobulin-like domains and interact with a dissociation constant of 2.5 microM and koff 0.8 s(-1), typical of many leukocyte protein membrane interactions. Pervanandate treatment of macrophages showed that OX2R could be phosphorylated on tyrosine residues. Blockade of the OX2-OX2R interaction with an OX2R mAb exacerbated the disease model experimental allergic encephalomyelitis. These data, together with data from an OX2-deficient mouse (R. M. Hoek et al., submitted), suggest that myeloid function can be controlled in a tissue-specific manner by the OX2-OX2R interaction.

Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores.

In this work, we have used spores of Bacillus subtilis that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0. 5%), and a pasteurization step (90 degrees C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the L-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate on L-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression.

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.

CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRPalpha 1.

The rat OX41 antigen is a cell surface protein containing three immunoglobulin superfamily domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIM). It is a homologue of the human signal-regulatory protein (SIRP) also known as SHPS-1, BIT or MFR. Cell activation-induced phosphorylation of the intracellular ITIM motifs induces association with the tyrosine phosphatases SHP-1 and SHP-2. To identify the physiological OX41 ligand, recombinant OX41-CD4d3+4 fusion protein was coupled to fluorescent beads to produce a multivalent cell binding reagent. The OX41-CD4d3+4 beads bound to thymocytes and concanavalin A-stimulated splenocytes. This interaction was blocked by the monoclonal antibody (mAb) OX101. Affinity chromatography with OX101 mAb and peptide sequencing revealed the rat SIRP ligand to be CD47 (integrin-associated protein). A direct interaction between human SIRP and human CD47 was demonstrated using purified recombinant proteins and surface plasmon resonance ruling out the involvement of other proteins known to be associated with CD47. The affinity of the SIRP/CD47 interaction was K(d) approximately 8 microM at 37 degrees C with a k(off )>/=2.1 s(-1). The membrane-distal SIRP V-like domain was sufficient for binding to CD47.

Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity.

Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

In vitro resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein is associated with alterations in cytoplasmic membrane fluidity.

Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729-732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169-3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293-3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1(r)) strains and their genetically related, tPMP-1-susceptible (tPMP-1(s)) counterpart strains. The tPMP-1(r) strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1(r) strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1(s) counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1(r) strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.

The TetA(K) tetracycline/H(+) antiporter from Staphylococcus aureus: mutagenesis and functional analysis of motif C.

Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from Staphylococcus aureus; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H(+) activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.

The role of microbiological testing in systems for assuring the safety of beef.

The use of microbiological testing in systems for assuring the safety of beef was considered at a meeting arranged by the International Livestock Educational Foundation as part of the International Livestock Congress, TX, USA, during February, 2000. The 11 invited participants from industry and government research organizations concurred in concluding that microbiological testing is necessary for the implementation and maintenance of effective Hazard Analysis Critical Control Point (HACCP) systems, which are the only means of assuring the microbiological safety of beef; that microbiological testing for HACCP purposes must involve the enumeration of indicator organisms rather than the detection of pathogens; that the efficacy of process control should be assessed against performance criteria and food safety objectives that refer to the numbers of indicator organisms in product; that sampling procedures should allow indicator organisms to be enumerated at very low numbers; and that food safety objectives and microbiological criteria are better related to variables, rather than attributes sampling plans.